HIV nef targeted ribozymes

ABSTRACT

An enzymatic nucleic acid molecule which cleaves an immunodeficiency virus RNA in a gene required for viral replication, e.g., the nef or tat gene regions.

This is a continuation of application Ser. No. 08/271,880 filed Jul. 7,1994, now U.S. Pat. No. 5,693,575 hereby incorporated by reference inits totality (including drawings), which is a continuation-in-part ofDraper, U.S. Ser. No. 07/882,886, filed May 14, 1992, now abandoned, andU.S. Ser. No. 08/103,423, filed Aug. 6, 1993, now abandoned, bothentitled "Method and Reagent for Inhibiting Human Immunodeficiency VirusReplication," assigned to the same assignee as the present application,the whole of which (including drawings) are hereby incorporated byreference herein and are abandoned in favor of the present application.

BACKGROUND OF THE INVENTION

This invention relates to the use of ribozymes as inhibitors of humanimmunodeficiency virus (HIV) replication, and in particular, theinhibition of HIV-1 replication. See e.g., Draper et al., PCT/WO93/23569hereby incorporated by reference.

Acquired immunodeficiency syndrome (AIDS) is thought to be caused byinfection with the virus HIV-1. At present, it is treated byadministration of the drug azidothymidine (AZT), which is thought toslow the progress of, but not cure, the disease. AZT resistant strainsof HIV-1 are found to develop after a year of treatment. In somepatients AZT has limited efficacy and may be found intolerable. Morerecently, drugs such as dideoxyinosine (DDI) and dideoxycytidine (DDC)have been tested as treatments for AIDS. None of these compounds reducethe viral load in patients, but they do treat the disease symptoms.

The following is a discussion of relevant art, none of which is admittedto be prior art to the pending claims. Rossi et al., 8 Aids Research andHuman Retroviruses 183, 1992, provide a review of the use of ribozymesas anti-HIV-1 therapeutic agents. They state:

An emerging strategy in the treatment of viral infections is the use ofantisense DNA or RNA to pair with, and block expression of viraltranscripts. RNA, in addition to being an informational molecule, canalso possess enzymatic activity. Thus, by combining anti-sense andenzymatic functions into a single transcript, it is now possible todesign catalytic RNAs, or ribozymes, which can specifically pair withvirtually any viral RNA, and cleave the phosphodiester backbone at aspecified location, thereby functionally inactivating the viral RNA. Incarrying out this cleavage, the ribozyme is not itself altered, and isthus capable of recycling and cleaving other molecules, making it a trueenzyme. There are several different catalytic motifs which possessenzymatic activity, and each one of these can be incorporated into anenzymatic antisense with site-specific cleavage capabilities.

Rossi et al. also state that studies have demonstrated that a hammerheadribozyme targeted to the gag gene RNA in the vicinity of thetranslational initiation codon is capable of specifically cleaving thattarget in a complex milieux of total cellular RNA. With reference toidentification of ribozyme targets in HIV-1 they state that mRNAs forthe two regulatory proteins tat and rev are clearly targets of choice,and that they are examining potential ribozyme cleavage sites in the tatmRNA, as well as in the exon shared by tat and rev. In addition, theystate:

A rational approach to the problem of target selection involves thefollowing criteria. First, one should select a functionally importanttarget, such as tat, rev, int, psi (packaging site), or the tRNA^(lys)priming site. Once a gene or target region has been decided upon, thenucleotide sequence should be assessed for strong conservation ofsequence among the various isolates. Within these conserved regions, thepotential cleavage sites, preferably G UC or GUA (others will suffice,but appear to be less efficiently cleaved) should be chosen. The regionshould be examined for potential secondary structures, and then the mostpromising sites chosen. Finally, before testing the ribozyme in cellculture, it is advisable to carry out a series of in vitro cleavagereactions (preferably kinetic analyses) using long (at least 100nucleotides in length) substrates to verify that the chosen sites aretruly structurally favorable for cleavage. [Citation omitted.]

Rossi et al. further state that a target which deserves furtherconsideration and testing as a potential ribozyme cleavage site is theviral packaging signal or psi sequence.

Sioud and Drlica, 88 Proc. Natl. Acad. Sci. USA 7303, 1991 describeribozymes designed to cleave the integrase gene of HIV. They state thatwhen the ribozyme is transcribed from a plasmid in E. coli it leads todestruction of the integrase RNA and complete blockage of integraseprotein synthesis. They state that the HIV-1 integrase gene may be auseful target for therapeutic ribozymes.

Heidenreich and Eckstein, 267 Journal of Biological Chemistry 1904,1992, describe three ribozymes targeted to different sites on the longterminal repeat (LTR) RNA of HIV-1. They also describe the influence ofchemical modifications within the ribozyme on the cleavage of the LTRRNA, including 2'-Fluorocytidine substitutions and phosphorothioateinternucleotidic linkages.

Weerasinghe et al., 65 Journal of Virology 5531, 1991, describeribozymes designed against a conserved region within the 5' leadersequence of HIV-1 RNA.

Chang et al., 2 Clinical Biotechnoloqy 23, 1990, describe ribozymesdesigned to target two different sites in the HIV-1 gag gene, and asingle site in the viral 5'-LTR region.

Lorentzen et al., 5 Virus Genes 17, 1991, describe a ribozyme targetedto the virion infectivity factor (vif) of HIV-1.

Sarver et al., 247 Science 1222, 1990, describe ribozymes in thehammerhead family targeted to HIV-1 gag transcripts. They state thatcells challenged with HIV-1 showed a substantial reduction in the levelof HIV-1 gag RNA relative to that in nonribozyme-expressing cells, andthat the reduction in gag RNA was reflected by a reduction in antigenp24 levels. They state that the results suggest the feasibility ofdeveloping ribozymes as therapeutic agents against human pathogens suchas HIV-1.

Hampel et al., "RNA Catalyst for Cleaving Specific RNA Sequences," filedSep. 20, 1989, which is a continuation-in-part of U.S. Ser. No.07/247,100, filed Sep. 20, 1988, describe hairpin ribozymes, andprovides an example of such a ribozyme apparently specific to the gaggene of HIV-1. Hampel and Tritz, 28 Biochemistry 4929, 1989 and Hampelet al., 18 Nucleic Acids Research 299, 1990 also describe hairpincatalytic RNA models and state that one target site is the tat gene inHIV-1.

Goldberg et al., WO 91/04319 and Robertson and Goldberg WO 91/04324,describe ribozymes expressed within a hepatitis delta vector and statethat the genome of the delta virus may carry a ribozyme against the envor gag mRNA of HIV. Rossi et al., WO 91/03162, describe chimeric DNA-RNAcatalytic sequences used to cleave HIV-1 gag transcript or the 5' LTRsplice site.

Ojwang et al., 89 Proc. Natl. Acad. Sci. USA 10, 802, 1992 and Yu etal., 90 Proc. Natl. Acad. Sci. USA 6340, 1993 describe a hairpinribozyme allegedly able to inhibit HIV-1 expression. Joseph and Burke268 J. Biol. Chem. 24, 515 1993, describe optimization of an anti-HIVhairpin ribozyme. Dropulic et al., 66 J. Virology 1432, 1992 describe aU5 ribozyme which cleaves at nucleoside +115 in HIV-1 RNA.

Other related art includes Rossi et al., U.S. Pat. Nos. 5,144,019 and5,149,796; Altman et al., U.S. Pat. No. 5,168,053; Zaia et al., 660 Ann.N.Y. Acad. Sci. 95, 1992; Guatelli et al., 16E J. Cell Biochem. 79,1992; Jeang et al., 267 J. Biol. Chem. 17891, 1992; Dropulic et al., 66J. Virol. 1432, 1992; Lisziewicz et al., International Publication WO91/10453; International Publication WO 91/15500; Rossi et al., 14A J.Cell Biochem. D428, 1990; and "The Papovaviridae", Ed. Salzman et al.,Vol. 2, The Viruses, Plenum Press, NY 1987.

Ribozymes are RNA molecules having an enzymatic activity which is ableto repeatedly cleave other separate RNA molecules in a nucleotide basesequence specific manner. Such enzymatic RNA molecules can be targetedto virtually any RNA transcript, and efficient cleavage has beenachieved in vitro. Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987;Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988;and Jefferies et al., 17 Nucleic Acids Research 1371, 1989.

Ribozymes act by first binding to a target RNA. Such binding occursthrough the target RNA binding portion of a ribozyme which is held inclose proximity to an enzymatic portion of the RNA which acts to cleavethe target RNA. Thus, the ribozyme first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After a ribozyme has bound and cleavedits RNA target it is released from that RNA to search for another targetand can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over othertechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the effective concentration of ribozyme necessary to effect atherapeutic treatment is lower than that of an antisenseoligonucleotide. This advantage reflects the ability of the ribozyme toact enzymatically. Thus, a single ribozyme molecule is able to cleavemany molecules of target RNA. In addition, the ribozyme is a highlyspecific inhibitor, with the specificity of inhibition depending notonly on the base pairing mechanism of. binding, but also on themechanism by which the molecule inhibits the expression of the RNA towhich it binds. That is, the inhibition is caused by cleavage of the RNAtarget and so specificity is defined as the ratio of the rate ofcleavage of the targeted RNA over the rate of cleavage of non-targetedRNA. This cleavage mechanism is dependent upon factors additional tothose involved in base pairing. Thus, it is thought that the specificityof action of a ribozyme is greater than that of antisenseoligonucleotide binding the same RNA site.

SUMMARY OF THE INVENTION

The invention features novel enzymatic RNA molecules, or ribozymes, andmethods for their use for inhibiting immunodeficiency virus replication,e.g., HIV-1, HIV-2 and related viruses including FIV-1 and SIV-1. Suchribozymes can be used in a method for treatment of diseases caused bythese related viruses in man and other animals. The invention alsofeatures cleavage of the RNA of these viruses by use of ribozymes. Inparticular, the ribozyme molecules described are targeted to the LTR,nef, vif, tat and rev viral genes or regions. These genes are known inthe art, see, e.g., Matsukura et al., 86 Proc. Natl. Acad. Sci. USA4244, 1989; Cheng-Mayer et al., 246 Science 1629, 1989; Viscidi et al.,246 Science 1606, 1989; Malim et al., 86 Proc. Natl. Acad. Sci. USA8222, 1989; Terwilliger et al., 88 Proc. Natl. Acad. Sci. USA 10971,1991; and Bartel et al., 67 Cell 529, 1991, and FIGS. 2A and 2B.

Thus, in a first aspect, the invention features an enzymatic RNAmolecule (or ribozyme) which cleaves HIV-1 RNA, or its equivalent,regions required for viral replication, e.g., protein synthesis, e.g.,the vif, nef, tat or rev gene regions, or at structures known toregulate viral gene expression, e.g., tar, rre or 3'-LTR regions.

By "enzymatic RNA molecule" it is meant an RNA molecule which hascomplementarity in a substrate binding region to a specified genetarget, and also has an enzymatic activity which is active tospecifically cleave RNA in that target. That is, the enzymatic RNAmolecule is able to intermolecularly cleave RNA and thereby inactivate atarget RNA molecule. This complementarity functions to allow sufficienthybridization of the enzymatic RNA molecule to the target RNA to allowthe cleavage to occur. One hundred percent complementarity is preferred,but complementarity as low as 50-75% may also be useful in thisinvention. By "equivalent" RNA to HIV-1 is meant to include thosenaturally occurring RNA molecules associated with immunodeficiencydiseases in various animals, including humans, felines, and simians.These viral RNAs have similar structures and equivalent genes to eachother, including the vif, nef, tat and rev genes.

By "gene" is meant to refer to either the protein coding regions of thecognate mRNA, HIV genome, proviral genome or any regulatory regions inthe RNA which regulate synthesis of the protein or stability of themRNA.

In preferred embodiments, the enzymatic RNA molecule is formed in ahammerhead motif, but may also be formed in the motif of a hairpin,hepatitis delta virus, group I intron or RNaseP-like RNA (in associationwith an RNA guide sequence). Examples of such hammerhead motifs aredescribed by Rossi et al. (see citations above), of hairpin motifs byHampel et al. (see citations above), and an example of the hepatitisdelta virus motif is described by Perrotta and Been, 31 Biochemistry 16,1992; of the RNaseP motif by Guerrier-Takada, et al., 35 Cell 849, 1983;and of the group I intron by Cech et al., U.S. Pat. No. 4,987,071. Thesespecific motifs are not limiting in the invention and those skilled inthe art will recognize that all that is important in an enzymatic RNAmolecule of this invention is that it has a specific substrate bindingsite which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule.

In particularly preferred embodiments, the RNA which is cleaved in HIV-1RNA is selected from one or more of the following sequences:

Sequence taken from the HIVPCV12 sequence in the Los Alamos HumanRetrovirus and AIDS database. The sequence folded began at nucleotidenumber one of the 2.3 kb subgenomic mRNA. This region includes thecoding regions for the vif, vpr, vpu, tat, rev, and nef gene products.

    __________________________________________________________________________    Nucleotide Sequence                                                           Number                                                                        __________________________________________________________________________                                  SEQ.ID.NO.                                        13 AGAAGAAAAGCAAAGAUCAUUAGGGAUUAUGGAAAACAGA                                                               ID.NO.01                                         108 AGUUUAGUAAAACAC                                                                                        ID.NO.02                                         121 CCAUAUGUAUAUUUC                                                                                        ID.NO.03                                         198 UCAGAAGUACACAUC                                                                                        ID.NO.04                                         228 AGAUUGGUAGUAANA                                                                                        ID.NO.05                                         235 AAUAACAACAUAUUGG                                                                                       ID.NO.06                                         246 AUUGGGGUCUGCAUA                                                                                        ID.NO.07                                         258 AUACAGGAGAAAGAGACUGGCAUUUGGG                                                                                                       ID.NO.08             280 AUCUGGGUCAGGGAGUCUCCAUA                                   ID.NO.09        311 AAAAAGAGAUAUAGCACACAAGUAGACCCU                                                                                                   ID.NO.10               439 UGAAUAUCAAGCAGGACAUAACAAGGUAGGAUCUCUACAAUA                                                                           ID.NO.11                           468 AUACUUGGCACUAGCAGCAUUAAUAACACCAAAAAAGAUAAAGC                                                                       ID.NO.12                             601 CACAAUGAAUGGACACUAG                                                                                    ID.NO.13                                         644 AAGCUGUUAGA                                                                                             ID.NO.14                                        683 UAGGGCAACAUAUCUAUGAAACUUA                                                                                                             ID.NO.15          733 GCCAUAAUAAGAA                                                                                          ID.NO.16                                         800 AUAGGCGUUAC                                                                                             ID.NO.17                                        828 GAAAUGGAGCC                                                                                             ID.NO.18                                        844 AUCCUAGACUAGAGC                                                                                        ID.NO.19                                         875 AAGUCAGCCUAAAA                                                                                         ID.NO.20                                         894 UGUACCAAUUGCUAUUGUAAAAAGUG                                                                                                           ID.NO.21           925 UUCAUUGCCAAG                                                                                           ID.NO.22                                         936 GUUUGUUUCAUAACAAAAGCCUUAGGCAUCUCCUAUGGCAGGAA                                                                       ID.NO.23                             988 GACAGCGACGAAGAG                                                                                        ID.NO.24                                         998 AAGACCUCCUCAAG                                                                                         ID.NO.25                                        1011 GGCAGUCAGACUCAUCAAGUUUCUCU                                                                                                           ID.NO.26          1037 AUCAAAGCAAC                                                                                             ID.NO.27                                       1053 UCCCAAUCCCGAGGGGACCCGACAGGCCCGAAGGAAUAGAAGAA                                                                       ID.NO.28                            1126 CAUUCGAUUAGUGAA                                                                                        ID.NO.29                                        1162 GGACGAUCUGCGGAGCCUGUGC                                     ID.NO.30      1260 GGGAAGCCCUCAAAUAUUGGUGGAAUCUC                                                                                                     ID.NO.31             1314 AGAAUAGUGCUG                                                                                           ID.NO.32                                        1339 UGCCACAGCUAUAGCA                                                                                       ID.NO.33                                        1383 AAGUAGUACAAGAAGCUUAUAGA                                   ID.NO.34       1419 UACCUAGAAGAAUAAGACAGGGCUUGGAAAGGAU                                                                                           ID.NO.35                  1476 UGGUCAAAAAGUAG                                                                                         ID.NO.36                                        1517 AAGAAUGAGACGAGCUGAGCCA                                     ID.NO.37      1557 GGAGCAGUAUCUCGA                                                                                        ID.NO.38                                        1568 AGACCUAGAAAAACAUGGAGCAAUCACA                                                                                                       ID.NO.39            1630 CCUGGCUAGAAGCACAAGAGGAGGAGAAGGUGGG                                                                                           ID.NO.40                  1674 ACACCUCAGGUACCUUUAAGACCAAUGACUUACAAG                                                                                       ID.NO.41                    1710 GCAGCUGUAGAUCUUAGCCACUUUUUAAAAGAAAAGGGGG                                                                               ID.NO.42                        1747 GGGGGACUGGAAGGG                                                                                        ID.NO.43                                        1760 GCUAAUUCACUCCCAACGA                                                                                    ID.NO.44                                        1779 AGACAAGAUAUCCUUGAUCUGUGGAUCUACCACA                                                                                           ID.NO.45                  1831 AUUGGCAGAACUACACACCAGGAC                                 ID.NO.46        1861 UCAGAUAUCCA                                                                                             ID.NO.47                                       1894 AAGCUAGUACCAGUU                                                                                        ID.NO.48                                        1941 GAGAACACCAGCUU                                                                                         ID.NO.49                                        1960 ACCCUGUGAGCCUGCAUGGAAUGGAUGAC                                                                                                     ID.NO.50             2008 AGUGGAGGUUUGACAGCCGC                                                                                   ID.NO.51                                        2065 AGUACUUCAAGAACUGCUGAUAUCGAGCUUGCUACAAGGGAC                                                                           ID.NO.52                          2188 CUGCUUUUUGCCUGUAC                                                                                      ID.NO.53                                        2228 UCUGAGCCUGGGAGCUC                                                                                      ID.NO.54                                        2281 UAAAGCUUGCC                                                                                             ID.NO.55                                       3'40 LTR:                                                                          UGCCUGUAGAUCCUAGAC                                                                                     ID.NO.56                                             AGCAUCCAGGAAGUCAGCC                                                                                    ID.NO.57                                        nef gene:                                                                       8-23    CAAGUGGUCAAAANG                                                                                   ID.NO.58                                         93-107   GGAGCAGUAUCUCAA                                                                                   ID.NO.59                                        214-229   CCNCAGGUACCUUUA                                                                                   ID.NO.60                                        283-297   GGGGGACUGGAUGGG                                                                                   ID.NO.61                                        vif gene:                                                                      80-95    CCAUAUGUAUGUUUC                                                                                   ID.NO.62                                        187-201   AGACUGGUAAUAANA                                                                                   ID.NO.63                                        239-253   AUCUGGGUCAGGGAG                                                                                   ID.NO.64                                                  AUUUGGGUCAGGGAG                                                                                   ID.NO.65                                        247-261   CAGGGAGUCUCCAUA                                                                                   ID.NO.66                                        286-300   ACACAAGUAGACCCU                                                                                   ID.NO.67                                        418-432   AACAAGGUAGGAUCU                                                     __________________________________________________________________________                                  ID.NO.68                                    

In a second related aspect, the invention features a mammalian cellwhich includes an enzymatic RNA molecule as described above. Preferably,the mammalian cell is a human cell, for example, a T4 lymphocyte havinga CD4 receptor molecule on its cell surface.

In a third related aspect, the invention features an expression vectorwhich includes nucleic acid encoding the enzymatic RNA moleculesdescribed above, located in the vector, e.g., in a manner which allowsexpression of that enzymatic RNA molecule within a mammalian cell.

In a fourth related aspect, the invention features a method fortreatment of human immunodeficiency disease by administering to apatient an enzymatic RNA molecule which cleaves HIV-1 RNA or related RNAin the vif, nef, tat or rev gene regions.

In other related aspects, the invention features treatment of cats orsimians with ribozymes of this invention. Such ribozymes may beidentical to those able to cleave HIV-1 RNA, or may be modified totarget analogous locations in FIV and SIV virus RNAs.

The invention provides a class of chemical cleaving agents which exhibita high degree of specificity for the viral RNA of HIV-1 typevirus-infected cells. If desired, such ribozymes can be designed totarget equivalent single-stranded DNAs by methods known in the art. Theribozyme molecule is preferably targeted to a highly conserved sequenceregion of HIV-1 such that all strains of HIV-1 can be treated with asingle ribozyme. Such enzymatic RNA molecules can be deliveredexogenously or endogenously to infected cells. In the preferredhammerhead motif, the small size (less than 40 nucleotides, preferablybetween 32 and 36 nucleotides in length) of the molecule allows the costof treatment to be reduced compared to other ribozyme motifs.

The smallest ribozyme delivered for treatment of HIV infection reportedto date (by Rossi et al., 1992, supra) is an in vitro transcript havinga length of 142 nucleotides. Synthesis of ribozymes greater than 100nucleotides in length is very difficult using automated methods, and thetherapeutic cost of such molecules is prohibitive. Delivery of ribozymesby expression vectors is primarily feasible using only ex vivotreatments. This limits the utility of this approach. In this invention,an alternative approach uses smaller ribozyme motifs (e.g., of thehammerhead structure, shown generally in FIG. 1) and exogenous delivery.The simple structure of these molecules also increases the ability ofthe ribozyme to invade targeted regions of the mRNA structure. Thus,unlike the situation when the hammerhead structure is included withinlonger transcripts, there are no non-ribozyme flanking sequences tointerfere with correct folding of the ribozyme structure, or with itscomplementary binding of the ribozyme to the mRNA target region.

The enzymatic RNA molecules of this invention can be used to treat humanimmunodeficiency virus infections, including those caused by both HIV-1and HIV-2. Such treatment can also be extended to other related viruseswhich infect non-human primates including the simian and felineimmunodeficiency viruses. Infected animals can be treated at the time ofproductive infection. This timing of treatment will reduce viral loadsin infected cells and disable viral replication in any subsequent roundsof infection. This is possible because the ribozymes disable thosestructures required for successful initiation of viral proteinsynthesis.

The targets chosen in the present invention provide a distinct advantageover prior targets since they act not only at the time of viralabsorption or reverse transcription during infection, but also inlatently infected cells and in virally transformed cells. In addition,viral particles which are released during a first round of infection inthe presence of such ribozymes will still be immunogenic by virtue ofhaving their capsids intact. Thus, one method of this invention allowsthe creation of defective but immunogenic viral particles, and thus acontinued possibility of initiation of an immune response in a treatedanimal.

In addition, the enzymatic RNA molecules of this invention can be usedin vitro in a cell culture infected with HIV-1 viruses, or relatedviruses, to produce viral particles which have intact capsids anddefective genomic RNA. These particles can then be used for instigationof immune responses in a prophylactic manner, or as a treatment ofinfected animals.

The invention also features immunization preparations formed fromdefective HIV-1 particles (or related particles) created by a method ofthis invention, and methods for immunization or vaccination using thesedefective particles, e.g., with DNA or vectors encoding a ribozyme ofthis invention under the control of a suitable promoter.

Diagnostic Uses

Ribozymes of this invention may be used as diagnostic tools to examinegenetic drift and mutations of viruses within diseased cells. The closerelationship between ribozyme activity and the structure of the targetRNA allows the detection of mutations in any region of the moleculewhich alters the base-pairing and three-dimensional structure of thetarget RNA. By using multiple ribozymes described in this invention, onemay map nucleotide changes which are important to RNA structure andfunction in vitro, as well as in cells and tissues. Cleavage of targetRNAs with ribozymes may be used to inhibit gene expression and definethe role (essentially) of specified gene products in the progression ofdisease. In this manner, other genetic targets may be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combinational therapies (e.g., multiple ribozymes targeted todifferent genes, ribozymes coupled with known small molecule inhibitors,or intermittent treatment with combinations of ribozymes and/or otherchemical or biological molecules). Other in vitro uses of ribozymes ofthis invention are well known in the art, and include detection of thepresence of mRNA associated with an HIV-1 related condition. Such RNA isdetected by determining the presence of a cleavage product aftertreatment with a ribozyme using standard methodology.

In a specific example, ribozymes which can cleave only wild-type HIV ormutant forms of the target RNA are used for the assay. The firstribozyme is used to identify wild-type RNA present in the sample and thesecond ribozyme will be used to identify mutant RNA in the sample. Asreaction controls, synthetic substrates of both wild-type and mutant RNAwill be cleaved by both ribozymes to demonstrate the relative ribozymeefficiencies in the reactions and the absence of cleavage of the"non-targeted" RNA species. The cleavage products from the syntheticsubstrates will also serve to generate size markers for the analysis ofwild-type and mutant RNAs in the sample population. Thus each analysiswill require two ribozymes, two substrates and one unknown sample whichwill be combined into six reactions. The presence of cleavage productswill be determined using an RNAse protection assay so that full-lengthand cleavage fragments of each RNA can be analyzed in one lane of apolyacrylamide gel. It is not absolutely required to quantify theresults to gain insight into the expression of mutant RNAs and putativerisk of the desired phenotypic changes in target cells. The expressionof mRNA whose protein product is implicated in the development of thephenotype (i.e., HIV) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels will be adequate and will decreasethe cost of the initial diagnosis. Higher mutant form to wild-typeratios will be correlated with higher risk whether RNA levels arecompared qualitatively or quantitatively.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings

FIG. 1 is a diagrammatic representation of a hammerhead motif ribozymeshowing stems I, II and III (marked (I), (II) and (III) respectively)interacting with an HIV-1 target region. The 5' and 3' ends of bothribozyme and target are shown. Dashes indicate base-paired nucleotides.

FIGS. 2A and 2B are diagrammatic representations of the various genesand gene regions in HIV-1 and HIV-2.

FIGS. 3A-3C are diagrammatic representations of three ribozymes of thisinvention.

FIGS. 4A-4G are diagrammatic representations of chemically modifiedribozymes of this invention (solid circles indicate modified bases).Specifically, FIG. 4A is unmodified HCH-r37, FIG. 4B is Thio-substitutedHCH-r37S2, FIG. 4C is Thio-substituted HCH-r37S4, FIGS. 4D-are Thiosubstituted HCH-s37A-D.

FIG. 5 is a diagrammatic representation of a chemically modifiedribozyme of this invention (circled bases are modified with2'-O-methyl), specifically the ribozyme is 2'-O-methyl HCH-037A.

FIG. 6 is a graphical representation of ribozyme stability in Vero cellor HeLa cell extracts from cytoplasm, membrane, and nucleus.

FIGS. 7, 8 and 9 are diagrammatic representations of various hammerheadribozymes along with data on activity, and nuclease resistance.

FIG. 10 is a graphical representation of activity of variousLTR-targeted ribozymes.

FIG. 11 is a graphical representation of activity of ribozymes ofvarious arm lengths.

FIG. 12 is a graphical representation of ribozymes with different sugarmodifications.

FIG. 13 is a graphical representation of activity of variousTAT-targeted ribozymes.

FIG. 14 is a diagrammatic representation of a hammerhead ribozymeshowing base numbering. Each N and N' can be the same or different.

FIG. 15 is a diagrammatic representation of two hairpin ribozymes activeon HIV RNA.

Target Sites

The genome of HIV-1 is subject to rapid genetic drift by virtue of itsRNA content and the nature of errors in reverse transcription. Thoseregions (genes) of the genome which are essential for virus replication,however, are expected to maintain a constant sequence (i.e., areconserved) over extensive periods of time. These regions are preferredtarget sites in this invention since they are more likely to beconserved between different types or strains of immunodeficiencyviruses, and thus only one ribozyme is needed to destroy all suchviruses. Thus, one ribozyme may be used to target all HIV-1 virus, aswell as all HIV-2, SIV and FIV viruses. We have selected several suchgenes of HIV-1, and examined their nucleotide sequences for the presenceof conserved regions which may be cleaved by ribozymes targeted to thoseregions. Two genes analyzed in detail are the vif and nef genes; thetat, rev and other genes noted above can be analyzed in a manner similarto that described below. Nucleotide sequences were acquired from the LosAlamos HIV gene bank.

Ribozymes targeting selected regions of the HIV genome are chosen tocleave the target RNA in a manner which inhibits translation of the RNA.Genes are selected such that inhibition of translation will inhibitviral replication, e.g., by inhibiting protein synthesis. Selection ofeffective target sites within these critical regions of HIV-1 RNAentails testing the accessibility of the target RNA to hybridizationwith various oligonucleotide probes. These studies can be performedusing RNA probes and assaying accessibility by cleaving the hybridmolecule with RNaseH (see below). Alternatively, such a study can useribozyme probes designed from secondary structure predictions of theRNAs, and assaying cleavage products by polyacrylamide gelelectrophoresis (PAGE), to detect the presence of cleaved and uncleavedmolecules.

The following is but one example of a method by which suitable targetsites can be identified and is not limiting in this invention.Generally, the method involves identifying potential cleavage sites fora hammerhead ribozyme, and then testing each of these sites to determinetheir suitability as targets by ensuring that secondary structureformation is minimal.

The HIV-1 genomic sequences of the Los Alamos data bank were compared inthe regions encoding the vif and nef genes. Fifteen putative ribozymecleavage sites were found to be highly conserved between the 11 strainsof virus sequence. These sites represent the preferable sites forhammerhead ribozyme cleavage within these two target RNAS. Two of thenef gene sites overlap regions within the 3'-LTR of the HIV-1 genome,which represents another target of potential therapeutic value. All ofthe nef targets are present in all known HIV-1 mRNAs and may representtargets for cleavage which would disrupt 3' terminal control regions ofthe mRNA which may be required for efficient translation or export ofthe RNAs. In a similar manner, a number of the vif target sites arepresent in the pol, tat and vpr mRNAs (see FIG. 2).

Short RNA substrates corresponding to each of the vif and nef gene siteswere designed. Each substrate was composed of two to three nucleotidesat the 5' and 3' ends that would not base pair with a correspondingribozyme recognition region. The unpaired regions flanked a centralregion of 12-14 nucleotides to which complementary arms in the ribozymewere designed.

The structure of each substrate sequence was predicted using a standardcommercially available PC fold computer program. Sequences which gave apositive free energy of binding were accepted. Sequences which gave anegative free energy were modified by trimming one or two bases fromeach of the ends. If the modified sequences were still predicted to havea strong secondary structure, they were rejected.

After substrates were chosen, ribozymes were designed to each of the RNAsubstrates. Ribozyme folding was also analyzed using PC fold.

Ribozyme molecules were sought which formed hammerhead motif stem II(see FIG. 1) regions and contained flanking arms which were devoid ofintramolecular base pairing. Often the ribozymes were modified bytrimming a base from the ends of the ribozyme, or by introducingadditional base pairs in stem II to achieve the desired fold. Ribozymeswith incorrect folding were rejected. After substrate/ribozyme pairswere found to contain correct intramolecular structures, the moleculeswere folded together to predict intermolecular interactions. A schematicrepresentation of a ribozyme with its coordinate base pairing to itscognate target sequence is shown in FIG. 1.

Using such analyses, the following predictions of effective target sitesin the vif and nef genes of the HIV genome, based upon computergenerated sequence comparisons, were obtained (see Table 1). The targetsequence is listed first with the 5'-most nucleotide number, forreference. Bases in parentheses are alternative bases in the conservedpatterns.

                                      TABLE 1                                     __________________________________________________________________________    Base numbers                                                                                RNA Target sequence                                             __________________________________________________________________________            nef gene                                                                8-23                   CAAGUGGU C AAAANG                                     93-107                      GGAGCAGU A UCUCGA                                                                       (A)                                    214-229             CCNCAGGU A CCUUUA                                         283-297                    (ALSO 3'40 LTR TARGET)                                                                                      (U)                  430-444                    (ALSO 3'40 LTR TARGET)                                                vif gene                                                    67-81                        AGUUUAGU A AAACAC                                80-95                        CCAUAUGU A UAUUUC                                                                                       (G)                   157-171                   UCAGAAGU A CACAUC                                   187-201                   AGAUUGGU A GUAANA                                                                                  (C)     (A)                    205-220                     AUUGGGGU C UGCAUA                                 239-253                   AUCUGGGU C AGGGAG                                                                                 (U)                             247-261                          CAGGGAGU C UCCAUA                            286-300                         ACACAAGU A GACCCU                             418-432                         AACAAGGU A GGAUCU                             __________________________________________________________________________

Those targets thought to be useful as ribozyme targets can be tested todetermine accessibility to nucleic acid probes in a ribonuciease H assay(see below) This assay provides a quick test of the use of the targetsite without requiring synthesis of a ribozyme. It can be used to screenfor sites most suited for ribozyme attack.

Synthesis of Ribozymes

Ribozymes useful in this invention can be produced by gene transcriptionas described by Cech, supra, or by chemical synthesis. Chemicalsynthesis of RNA is similar to that for DNA synthesis. The additional2'-OH group in RNA, however, requires a different protecting groupstrategy to deal with selective 3'-5' internucleotide bond formation,and with RNA susceptibility to degradation in the presence of bases. Therecently developed method of RNA synthesis utilizing thet-butyldimethylsilyl group for the protection of the 2' hydroxyl is themost reliable method for synthesis of ribozymes. The method reproduciblyyields RNA with the correct 3'-5' internucleotide linkages, with averagecoupling yields in excess of 99%, and requires only a two-stepdeprotection of the polymer.

A method, based upon H-phosphonate chemistry exhibits a relatively lowercoupling efficiency than a method based upon phosphoramidite chemistry.This is a problem for synthesis of DNA as well. A promising approach toscale-up of automatic oligonucleotide synthesis has been describedrecently for the H-phosphonates. A combination of a proper coupling timeand additional capping of "failure" sequences gave high yields in thesynthesis of oligodeoxynucleotides in scales in the range of 14 μmoleswith as little as 2 equivalents of a monomer in the coupling step.Another alternative approach is to use soluble polymeric supports (e.g.,polyethylene glycols), instead of the conventional solid supports. Thismethod can yield short oligonucleotides in hundred milligram quantitiesper batch utilizing about 3 equivalents of a monomer in a coupling step.

Various modifications to ribozyme structure can be made to enhance theutility of ribozymes. Such modifications will enhance shelf-life,half-life in vitro, stability, and ease of introduction of suchribozymes to the target site, e.g., to enhance penetration of cellularmembranes, and confer the ability to recognize and bind to targetedcells.

Exogenous delivery of ribozymes benefits from chemical modification ofthe backbone, e.g., by the overall negative charge of the ribozymemolecule being reduced to facilitate diffusion across the cell membrane.The present strategies for reducing the oligonucleotide charge include:modification of internucleotide linkages by methylphosphonates, use ofphosphoramidites, linking oligonucleotides to positively chargedmolecules, and creating complex packages composed of oligonucleotides,lipids and specific receptors or effectors for targeted cells. Examplesof such modifications include sulfur-containing ribozymes containingphosphorothioates and phosphorodithioates as internucleotide linkages inRNA. Synthesis of such sulfur-modified ribozymes is achieved by use ofthe sulfur-transfer reagent, ³ H-1,2-benzenedithiol-3-one 1,1-dioxide.Ribozymes may also contain ribose modified ribonucleotides. Pyrimidineanalogues are prepared from uridine using a procedure employingdiethylamino sulphur trifluoride (DAST) as a starting reagent. Ribozymescan also be either electrostatically or covalently attached to polymericcations for the purpose of reducing charge. The polymer can be attachedto the ribozyme by simply converting the 3'-end to a ribonucleosidedialdehyde which is obtained by a periodate cleavage of the terminal 2',3'-cis diol system. Depending on the specific requirements for deliverysystems, other possible modifications may include different linker arms.containing carboxyl, amino or thiol functionalities. Yet furtherexamples include use of methylphosphonates and 2'-O-methylribose and 5'or 3' capping or blocking with m₇ GpppG or m₃ ²,2,7 GpppG.

For example, a kinased ribozyme is contacted with guanosine triphosphateand guanyltransferase to add a m³ G cap to the ribozyme. After suchsynthesis, the ribozyme can be gel purified using standard procedure. Toensure that the ribozyme has the desired activity, it may be tested withand without the 5' cap using standard procedures to assay both itsenzymatic activity and its stability.

Synthetic ribozymes, including those containing various modifiers, canbe purified by high pressure liquid chromatography (HPLC). Other liquidchromatography techniques, employing reverse phase columns and anionexchangers on silica and polymeric supports may also be used.

There follows an example of the synthesis of one ribozyme. A solid phasephosphoramidite chemistry was employed. Monomers used were2'-tert-butyl-dimethylsilyl cyanoethylphosphoramidites of uridine,N-benzoyl-cytosine, N-phenoxyacetyl adenosine, and guanosine (GlenResearch, Sterling, Va.).

Solid phase synthesis was carried out on either an ABI 394 or 380BDNA/RNA synthesizer using the standard protocol provided with eachmachine. The only exception was that the coupling step was increasedfrom 10 to 12 minutes. The phosphoramidite concentration was 0.1 M.Synthesis was done on a 1 μmole scale using a 1 μmole RNA reactioncolumn (Glen Research). The average coupling efficiencies were between97% and 98% for the 394 model, and between 97% and 99% for the 380Bmodel, as determined by a calorimetric measurement of the releasedtrityl cation.

After synthesis, the blocked ribozymes were cleaved from the solidsupport (e.g., CPG), and the bases and diphosphoester moiety deprotectedin a sterile vial by incubation in dry ethanolic ammonia (2 mL) at 55°C. for 16 hours. The reaction mixture was cooled on dry ice. Later, thecold liquid was transferred into a sterile screw cap vial andlyophilized.

To remove the 2'-tert-butyl-dimethylsilyl groups from the ribozyme, theobtained residue was suspended in 1 M tetra-n-butylammonium fluoride indry THF (TBAF), using a 20-fold excess of the reagent for every silylgroup, for 16 hours at ambient temperature (about 15-25° C.). Thereaction was quenched by adding an equal volume of sterile 1 Mtriethylamine acetate, pH 6.5. The sample was cooled and concentrated ona SpeedVac to half the initial volume.

The ribozymes were purified in two steps by HPLC on a C4 300 Å 5 μmDeltaPak column in an acetonitrile gradient.

The first step, or "trityl on" step, was a separation of5'-DMT-protected ribozyme(s) from failure sequences lacking a 5'-DMTgroup. Solvents used for this step were: A (0.1 M Triethylammoniumacetate, pH 6.8) and B (acetonitrile). The elution profile was: 20% Bfor 10 minutes, followed by a linear gradient of 20% B to 50% B over 50minutes, 50% B for 10 minutes, a linear gradient of 50% B to 100% B over10 minutes, and a linear gradient of 100% B to 0% B over 10 minutes.

The second step was a purification of a completely deblocked ribozyme bya treatment of 2% trifluoroacetic acid on a C4 300 Å 5 μm DeltaPakcolumn in an acetonitrile gradient. Solvents used for this second stepwere: A (0.1 M Triethylammonium acetate, pH 6.8) and B (80%acetonitrile, 0.1 M triethylammonium acetate, pH 6.8). The elutionprofile was: 5% B for 5 minutes, a linear gradient of 5% B to 15% B over60 minutes, 15% B for 10 minutes, and a linear gradient of 15% B to 0% Bover 10 minutes.

The fraction containing ribozyme, which is in the triethylammonium saltform, was cooled and lyophilized on a SpeedVac. Solid residue wasdissolved in a minimum amount of ethanol and ribozyme in sodium saltform was precipitated by addition of sodium perchlorate in acetone. (K⁺or Mg²⁺ salts can be produced in an equivalent manner.) The ribozyme wascollected by centrifugation, washed three times with acetone, andlyophilized.

Expression Vector

While synthetic ribozymes are preferred in this invention, thoseproduced by expression vectors can also be used. In designing a suitableribozyme expression vector the following factors are important toconsider. The final ribozyme must be kept as small as possible tominimize unwanted secondary structure within the ribozyme. A promoter(e.g., the human cytomegalovirus immediate early region (HCMV ie1)promoter) should be chosen to be a relatively strong promoter, andexpressible both in vitro and in vivo. Such a promoter should expressthe ribozyme at a level suitable to effect production of enough ribozymeto destroy a target RNA, but not at too high a level to prevent othercellular activities from occurring (unless cell death itself isdesired).

A hairpin at the 5' end of the ribozyme is useful to ensure that therequired transcription initiation sequence (GG or GGG or GGGAG) does notbind to some other part of the ribozyme and thus affect regulation ofthe transcription process. The 5' hairpin is also useful to protect theribozyme from 5'-3' exonucleases. A selected hairpin at the 3' end ofthe ribozyme gene is useful since it acts as a transcription terminationsignal, and as a protection from 3'-5' exonuclease activity. One exampleof a known termination signal is that present on the T7 RNA polymerasesystem. This signal is about 30 nucleotides in length. Other 3' hairpinsof shorter length can be used to provide good termination and RNAstability. Such hairpins can be inserted within the vector sequences toallow standard ribozymes to be placed in an appropriate orientation andexpressed with such sequences attached.

Poly(A) tails are also useful to protect the 3' end of the ribozyme.These can be provided by either including a poly(A) signal site in theexpression vector (to signal a cell to add the poly(A) tail in vivo), orby introducing a poly(A) sequence directly into the expression vector.In the first approach, the signal must be located to prevent unwantedsecondary structure formation with other parts of the ribozyme. In thesecond approach, the poly(A) stretch may reduce in size over time whenexpressed in vivo, and thus the vector may need to be checked over time.Care must be taken in addition of a poly(A) tail which binds poly(A)binding proteins which prevent the ribozyme from acting upon theirtarget sequence.

Ribozyme Testing

Once the desired ribozymes are selected, synthesized and purified, theyare tested in kinetic and other experiments to determine their utility.An example of such a procedure is provided below.

Preparation of Ribozyme

Crude synthetic ribozyme (typically 350 μg at a time) is purified byseparation on a 15% denaturing polyacrylamide gel (0.75 mm thick, 40 cmlong) and visualized by UV shadowing. Once excised, gel slicescontaining full length ribozyme are soaked in 5 ml gel elution buffer(0.5 M NH₄ OAc, 1 mM EDTA) overnight with shaking at 4° C. The eluent isdesalted over a C-18 matrix (Sep-Pak cartridges, Millipore, Milford,Mass.) and vacuum dried. The dried RNA is resuspended in 50-100 μl TE(TRIS 10 mM, EDTA 1 mM, pH 7.2). An aliquot of this solution is diluted100-fold into 1 ml TE, half of which is used to spectrophotometricallyquantitate the ribozyme solution. The concentration of this dilute stockis typically 150-800 nM. Purity of the ribozyme is confirmed by thepresence of a single band on a denaturing polyacrylamide gel.

A ribozyme may advantageously be synthesized in two or more portions.Each portion of a ribozyme will generally have only limited or noenzymatic activity, and the activity will increase substantially (by atleast 5-10 fold) when all portions are ligated (or otherwise juxtaposed)together. A specific example of hammerhead ribozyme synthesis isprovided below.

The method involves synthesis of two (or more) shorter "half" ribozymesand ligation of them together using T4 RNA ligase. For example, to makea 34 mer ribozyme, two 17 mers are synthesized, one is phosphorylated,and both are gel purified. These purified 17 mers are then annealed to aDNA splint strand complementary to the two 17 mers. This DNA splint hasa sequence designed to locate the two 17 mer portions with one end ofeach adjacent each other. The juxtaposed RNA molecules are then treatedwith T4 RNA ligase in the presence of ATP. Alternatively, the DNA splintstrand may be omitted from the ligation reaction if the complementarybinding affects favorable ligation of the two RNA molecules. The 34 merRNA so formed is then HPLC purified.

Preparation of Substrates

Approximately 10-30 pmoles of unpurified substrate is radioactively 5'end-labeled with T4 polynucleotide kinase using 25 pmoles of [γ-³² P]ATP. The entire labeling mix is separated on a 20% denaturingpolyacrylamide gel and visualized by autoradiography. The full lengthband is excised and soaked overnight at 4° C. in 100 μl of TE (10 mMTris-HCl pH 7.6, 0.1 mM EDTA).

Kinetic Reactions

For reactions using short substrates (between 8 and 16 bases) asubstrate solution is made 1× in assay buffer (75 mM Tris-HCl, pH 7.6;0.1 mM EDTA, 10 mM MgCl₂) such that the concentration of substrate isless than 1 nM. A ribozyme solution (typically 20 nM) is made 1× inassay buffer and four dilutions are made using 1× assay buffer. Fifteenμl of each ribozyme dilution (i.e., 20, 16, 12, 8 and 4 nM) is placed ina separate tube. These tubes and the substrate tube are pre-incubated at37° C. for at least five minutes.

The reaction is started by mixing 15 μl of substrate into each ribozymetube by rapid pipetting (note that final ribozyme concentrations are 10,8, 6, 4, 2 nM). Five μl aliquots are removed at 15 or 30 secondintervals and quenched with 5 μl stop solution (95% formamide, 20 mMEDTA xylene cyanol, and bromphenol blue dyes). Following the finalribozyme time point, an aliquot of the remaining substrate is removed asa zero ribozyme control.

The samples are separated on either 15% or 20% polyacrylamide gels. Eachgel is visualized and quantitated with an Ambis beta scanner (AmbisSystems, San Diego, Calif.).

For the most active ribozymes, kinetic analyses are performed insubstrate excess to determine K_(m) and K_(cat) values.

For kinetic reactions with long RNA substrates (greater than 15 bases inlength) the substrates are prepared by transcription using T7 RNApolymerase and defined templates containing a T7 promoter, and DNAencoding appropriate nucleotides of the HIV-1 RNA. The substratesolution is made 1× in assay buffer (75 mM Tris-HCl, pH 7.6; 0.1 mMEDTA; 10 mM MgCl₂) and contains 58 nanomolar concentration of the longRNA molecules. The reaction is started by addition of gel purifiedribozymes to 1 μM concentration. Aliquots are removed at 20, 40, 60, 80and 100 minutes, then quenched by the addition of 5 μl stop solution.Cleavage products are separated using denaturing PAGE. The bands arevisualized and quantitated with an Ambis beta scanner.

Kinetic Analysis

A simple reaction mechanism for ribozyme-mediated cleavage is: ##EQU1##where R=ribozyme, S=substrate, and P=products. The boxed step isimportant only in substrate excess. Because ribozyme concentration is inexcess over substrate concentration, the concentration of theribozyme-substrate complex ([R:S]) is constant over time except duringthe very brief time when the complex is being initially formed, i.e.,:##EQU2## where t=time, and thus:

    (R)(S)k.sub.1 =(RS)(k.sub.2 +k.sub.1).

The rate of the reaction is the rate of disappearance of substrate withtime: ##EQU3## Substituting these expressions: ##EQU4## Integrating thisexpression with respect to time yields: ##EQU5## where S₀ =initialsubstrate. Therefore, a plot of the negative log of fraction substrateuncut versus time (in minutes) yields a straight line with slope:##EQU6## where k_(obs) =observed rate constant. A plot of slope(k_(obs)) versus ribozyme concentration yields a straight line with aslope which is: ##EQU7##

Using these equations the data obtained from the kinetic experimentsprovides the necessary information to determine which ribozyme tested ismost useful, or active. Such ribozymes can be selected and tested in invivo or ex vivo systems.

Liposome Preparation

Lipid molecules were dissolved in a volatile organic solvent (CHCl₃,methanol, diethylether, ethanol, etc.). The organic solvent was removedby evaporation. The lipid was hydrated into suspension with 0.1×phosphate buffered saline (PBS), then freeze-thawed 3× using liquidnitrogen and incubation at room temperature. The suspension was extrudedsequentially through a 0.4 μm, 0.2 μm and 0.1 μm polycarbonate filtersat maximum pressure of 800 psi. The ribozyme was mixed with the extrudedliposome suspension and lyophilized to dryness. The lipid/ribozymepowder was rehydrated with water to one-tenth the original volume. Thesuspension was diluted to the minimum volume required for extrusion (0.4ml for 1.5 ml barrel and 1.5 ml for 10 ml barrel) with 1×PBS andre-extruded through 0.4 μm, 0.2 μm, 0.1 μm polycarbonate filters. Theliposome entrapped ribozyme was separated from untrapped ribozyme by gelfiltration chromatography (SEPHAROSE CL-4B, BIOGEL A5M). The liposomeextractions were pooled and sterilized by filtration through a 0.2 μmfilter. The free ribozyme was pooled and recovered by ethanolprecipitation. The liposome concentration was determined byincorporation of a radioactive lipid. The ribozyme concentration wasdetermined by labeling with ³². Rossi et al., 1992, supra (andreferences cited therein), describe other methods suitable forpreparation of liposomes.

In experiments with a liposome formulation composed of a synthetic lipidderivative disteraoyl-phosphatidylethylamidothioacetyl succinimide(DSPE-ATS) co-formulated with dipalmitoylphosphatidyl choline andcholesterol we observed uptake of 100 and 200 nm diameter liposomes withsimilar kinetics. The larger particles accommodated a larger number ofentrapped molecules, or larger molecular weight molecules, such as anexpression plasmid. These particles showed a linear relationship betweenthe lipid dose offered and the mean log fluorescence (calcein was usedto follow liposome uptake). No cytotoxicity was observed even with a 200μM dose. These liposomes are particularly useful for delivery to CD4cell populations.

In Vivo Assay

The efficacy of action of a chosen ribozyme may be tested in vivo by useof cell cultures sensitive to HIV-1 or a related virus, using standardprocedures. For example, monolayer cultures of HIV-sensitive cells aregrown by established procedures. Cells are grown in 6 or 96 well tissueculture plates. Prior to infection with HIV, cultures are treated for 3to 24 hours with ribozyme-containing liposomes. Cells are then rinsedwith phosphate buffered saline (PBS) and virus added at a multiplicityof 1-100 pfu/cell. After a one-hour adsorption, free virus is rinsedaway using PBS, and the cells are treated for three to five days withappropriate liposome preparations. Cells are then re-fed with freshmedium and re-incubated. Virus is harvested from cells into theoverlying medium. Cells are broken by three cycles of incubation at -70°C. and 37° C. for 30 minutes at each temperature, and viral titersdetermined by plaque assay using established procedures.

Ribonuclease Protection Assay

The accumulation of target mRNA in cells or the cleavage of the RNA byribozymes or RNaseH (in vitro or in vivo) can be quantified using anRNase protection assay.

In this method, antisense riboprobes are transcribed from template DNAusing T7 RNA polymerase (U.S. Biochemical) in 20 μl reactions containing1× transcription buffer (supplied by the manufacturer), 0.2 mM ATP, GTPand UTP, 1 U/μl pancreatic RNase inhibitor (Boehringer MannheimBiochemicals) and 200 μCi ³² P-labeled CTP (800 Ci/mmol, New EnglandNuclear) for 1 hour at 37° C. Template DNA is digested with 1 URNase-free DNaseI (U.S. Biochemical, Cleveland, Ohio) at 37° C. for 15minutes and unincorporated nucleotides removed by G-50 SEPHADEX spinchromatography.

In a manner similar to the transcription of antisense probe, the targetRNA can be transcribed in vitro using a suitable DNA template. Thetranscript is purified by standard methods and digested with ribozyme at37° C. according to methods described later.

Alternatively, virus-infected cells are harvested into 1 ml of PBS,transferred to a 1.5 ml EPPENDORF tube, pelleted for 30 seconds at lowspeed in a microcentrifuge, and lysed in 70 μl of hybridization buffer(4 M guanidine isothiocyanate, 0.1% sarcosyl, 25 mM sodium citrate, pH7.5). Cell lysate (45 μl) or defined amounts of in vitro transcript(also in hybridization buffer) is then combined with 5 μl ofhybridization buffer containing 5×10⁵ cpm of each antisense riboprobe in0.5 ml Eppendorf tubes, overlaid with 25 Ml mineral oil, andhybridization accomplished by heating overnight at 55° C. Thehybridization reactions are diluted into 0.5 ml RNase solution (20 U/mlRNaseA, 2 U/ml RNaseT1, 10 U/ml RNase-free DNaseI in 0.4 M NaCl), heatedfor 30 minutes at 37° C., and 10 μl of 20% SDS and 10 μl of Proteinase K(10 mg/ml) added, followed by an additional 30 minutes incubation at 37°C. Hybrids are partially purified by extraction with 0.5 ml of a 1:1mixture of phenol/chloroform; aqueous phases are combined with 0.5 mlisopropanol, and RNase-resistant hybrids pelleted for 10 minutes at roomtemperature (about 20° C.) in a microcentrifuge. Pellets are dissolvedin 10 μl loading buffer (95% formamide, 1×TBE, 0.1% bromophenol blue,0.1% xylene cylanol), heated to 95° C. for five minutes, cooled on ice,and analyzed on 4% polyacrylamide/7 M urea gels under denaturingconditions.

Ribozyme Stability

The chosen ribozyme can be tested to determine its stability, and thusits potential utility. Such a test can also be used to determine theeffect of various chemical modifications (e.g., addition of a poly(A)tail) on the ribozyme stability and thus aid selection of a more stableribozyme. For example, a reaction mixture contains 1 to 5 pmoles of 5'(kinased) and/or 3' labeled ribozyme, 15 μg of cytosolic extract and 2.5mM MgCl₂ in a total volume of 100 Ml. The reaction is incubated at 37°C. Eight μl aliquots are taken at timed intervals and mixed with 8 μl ofa stop mix (20 mM EDTA, 95% formamide). Samples are separated on a 15%acrylamide sequencing gel, exposed to film, and scanned with an Ambis.

A 3'-labeled ribozyme can be formed by incorporation of the ³² P-labeledcordycepin at the 3' OH using poly(A) polymerase. For example, thepoly(A) polymerase reaction contains 40 mM Tris, pH 8, 10 mM MgCl₂, 250mM NaCl, 2.5 mM MnCl₂ ; 3 μl P³² cordycepin, 500 Ci/mM; and 6 unitspoly(A) polymerase in a total volume of 50 μl. The reaction mixture isincubated for 30 minutes at 37° C.

Effect of Liposome Surface Modifications on Lymphocyte and MacroPhageUptake

Liposomes containing distearoylphosphatidyl ethanolamidomethylthioacetate can be prepared. The thiol group can be deprotected usinghydroxylamine and the reactive thiol can then be modified with thiolreactive groups to alter the surface properties of the liposomes.Reaction with N-ethylmaleimide leads to lymphocyte and macrophageuptake. This modification results in liposome uptake by CD4⁻ and CD4⁺lymphocytes.

Modification with iodoacetamide and iodoacetic acid were tested foruptake. Iodactamide modification showed that the liposomes were toxic inthat the lymphocyte cell number decreased with time. However, theacetate uptake was 5-fold greater than that observed for succinimide.Uptake was monitored-by both uptake of a ³ H-hexadecylcholesterol etherand an entrapped water soluble fluorophore, calcein. Both confirmed thatthe liposomes were being taken up by the cells over a 72 hour period.The uptake was assayed in lymphoblasts which had been stimulated for 72hours with phytohemagglutinin. The uptake curve was biphasic in thatwithin the first 8 hours, only cell surface binding was observed whereasafter 24 hours, uptake was linear for up to 72 hours. This was observedfor a 200 μM dose. The 100 μM lipid dose showed only cell surfacebinding.

Effect of Base Substitution Upon Ribozyme Activity

To determine which primary structural characteristics could changeribozyme cleavage of substrate, minor base changes can be made in thesubstrate cleavage region recognized by a specific ribozyme. Forexample, the substrate sequences can be changed at the central "C"nucleotide, changing the cleavage site from a GUC to a GUA motif. TheK_(cat) /K_(m) values for cleavage using each substrate are thenanalyzed to determine if such a change increases ribozyme cleavagerates. Similar experiments can be performed to address the effects ofchanging bases complementary to the ribozyme binding arms. Changespredicted to maintain strong binding to the complementary substrate arepreferred. Minor changes in nucleotide content can alterribozyme/substrate interactions in ways which are unpredictable basedupon binding strength alone. Structures in the catalytic core region ofthe ribozyme recognize trivial changes in either substrate structure orthe three dimensional structure of the ribozyme/substrate complex.

To begin optimizing ribozyme design, the cleavage rates of ribozymescontaining varied arm lengths, but targeted to the same length of shortRNA substrate can be tested. Minimal arm lengths are required andeffective cleavage varies with ribozyme/substrate combinations.

The cleavage activity of selected ribozymes can be assessed usingHIV-1-homologous substrates. The assays are performed in ribozyme excessand approximate K_(cat) /K_(min) values obtained. Comparison of valuesobtained with short and long substrates indicates utility in vivo of *aribozyme.

Intracellular Stability of Liposome-delivered Ribozymes

To test the stability of a chosen ribozyme in vivo the following test isuseful. Ribozymes are ³² P-end-labeled, entrapped in liposomes anddelivered to HIV-1 sensitive cells for three hours. The cells arefractionated and ribozyme is purified by phenol/chloroform extraction.Alternatively, cells (1×10⁷, T-175 flask) are scraped from the surfaceof the flask and washed twice with cold PBS. The cells are homogenizedby douncing 35 times in 4 ml of TSE (10 mM Tris, pH 7.4, 0.25 M Sucrose,1 mM EDTA). Nuclei are pelleted at 100×g for 10 minutes. Subcellularorganelles (the membrane fraction) are pelleted at 200,000×g for twohours using an SW60 rotor. The pellet is resuspended in 1 ml of H buffer(0.25 M Sucrose, 50 mM HEPES, pH 7.4). The supernatant contains thecytoplasmic fraction (in approximately 3.7 ml). The nuclear pellet isresuspended in 1 ml of 65% sucrose in TM (50 mM Tris, pH 7.4, 2.5 mMMgCl₂) and banded on a sucrose step gradient (1 ml nuclei in 65% sucroseTM, 1 ml 60% sucrose TM, 1 ml 55% sucrose TM, 50% sucrose TM, 300 μl 25%sucrose TM) for one hour at 37,000×g with an SW60 rotor. The nuclearband is harvested and diluted to 10% sucrose with TM buffer. Nuclei arepelleted at 37,000×g using an SW60 rotor for 15 minutes and the pelletresuspended in 1 ml of TM buffer. Aliquots are size fractionated ondenaturing polyacrylamide gels and the intracellular localizationdetermined. By comparison to the migration rate of newly synthesizedribozyme, the various fraction containing intact ribozyme can bedetermined.

To investigate modifications which would lengthen the half-life ofribozyme molecules intracellularly, the cells may be fractioned as aboveand the purity of each fraction assessed by assaying enzyme activityknown to exist in that fraction.

The various cell fractions are frozen at -70° C. and used to determinerelative nuclease resistances of modified ribozyme molecules. Ribozymemolecules may be synthesized with 5 phosphorothioate (ps), or2'-O-methyl (2'-OMe) modifications at each end of the molecule. Thesemolecules and a phosphodiester version of the ribozyme are end-labeledwith ³² P and ATP using T4 polynucleotide kinase. Equal concentrationsare added to the cell cytoplasmic extracts and aliquots of each taken at10 minute intervals. The samples are size fractionated by denaturingPAGE and relative rates of nuclease resistance analyzed by scanning thegel with an Ambis β-scanner. The results show whether the ribozymes aredigested by the cytoplasmic extract, and which versions are relativelymore nuclease resistant. Modified ribozymes generally maintain 80-90% ofthe catalytic activity of the native ribozyme when short RNA substratesare employed.

Unlabeled, 5' end-labeled or 3' end-labeled ribozymes can be used in theassays. These experiments can also be performed with human cell extractsto verify the observations.

HeLa (CD4⁺) and Vero cells are used by many investigators to screenantiviral chemicals in pre-clinical trials. Ribozymes and antisense DNAsdepend upon their nuclease stability to maintain therapeutic levels ininfected cells. To design chemical modifications which increase thenuclease stability of ribozymes, we have prepared nuclear, cytoplasmicand membranous lysates from various cell types and compared the relativespecific activity (units of nuclease/mg protein) and cation optima (Mg⁺²-, Mn⁺² -, Ca⁺² - or Zn⁺² -activation) of the nuclease populationswithin these fractions. Nuclease activities vary significantly among thecell fractions studied (Tables 2 and 3, and FIG. 6; +++ indicates highnuclease activity, ++ and + indicate lower levels, and - indicates nonuclease activity as tested by standard methodology), but the nucleasepopulation present in HeLa cells was dissimilar to that observed in bothmonocyte and lymphocyte preparations (Table 4, standard units are notedin the table for comparison only and are not reflective of a specificlevel of nuclease activity).

                  TABLE 2                                                         ______________________________________                                        RELATIVE ACTIVITY OF CYTOPLASMIC EXTRACTS                                                                 Divalent cation                                   Cell Type     Units/10 ug of protein                                                                      Requirement                                       ______________________________________                                        Vero          1             Mg.sup.++                                         HeLa          5.8           Zn.sup.++                                         Cervical Epithelial                                                                         4.7           Zn.sup.++                                         Monocyte enriched                                                                           12.9          none                                              T Lymphocytes 23.5          none                                              Keratinocytes 4.8           Zn.sup.++                                         ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Divalent cation effect on RNA degradative activity in HeLa fractions                     Divalent cation (1 mM)                                             HeLa cell fractions                                                                        MgCl.sub.2                                                                            MnCl.sub.2                                                                              CaCl.sub.2                                                                          ZnCl.sub.2                               ______________________________________                                        Cytoplasmic  +       +         +     +++                                      Membrane     +++     +++       +     -                                        Nuclear      +++     ++        +++   ++                                       ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Divalent cation effect on RNA degradative activity in Vero fraction                       Divalent Cation (1 mM)                                            Vero cell fractions                                                                         MgCl.sub.2                                                                            MnCl.sub.2                                                                              CaCl.sub.2                                                                          ZnCl.sub.2                              ______________________________________                                        Cytoplasmic   +++     +++       -     -                                       Membrane      +++     +++       -     -                                       Nuclear       +++     +++       ++    -                                       ______________________________________                                    

HeLa nucleases were maximally active in the presence of added Zn⁺²,while added cation did not enhance nuclease activities in monocyte andlymphocyte lysates. The relative nuclease levels in the monocyte andlymphocyte lysates were 2.2 and 4 times higher, respectively, than inactivated HeLa lysates.

Various chemical modifications to ribozymes were tested for theirability to increase nuclease activity while maintaining catalyticactivity of the ribozyme. FIG. 7 shows the effects of variousmodifications upon the catalytic activity of a selected ribozyme. Themodifications which decreased activity (1315, 1371, and 1285) weredropped from further analyses. Chemical modifications to ribozymesresulted in cell-specific patterns of ribozyme stability, and2'-O-methyl sugar substitution gave the best overall enhancement ofribozyme stability across lysates. Relative resistance of ribozymes todigestion by lymphocyte and monocyte cytoplasmic lysates are shown inFIG. 8. The digestion rates of the ribozymes are similar when theribozymes are labeled at either the 5'- or 3'-ends of the molecule,demonstrating that the degradation is not due to endogenous phosphataseactivity in the lysates.

Various reports have suggested that modifications to hammerhead motifribozymes in the binding arms (stems I and III) will give much enhancedprotection from nuclease digestion. As shown in FIG. 9, we observedifferent results. There appear to be nuclease resistant sites at thepositions 2.4, 2.5, 7, 8, 9, 10.4, and 13. The 2.4, 2.5 and 10.4position resistance may be specific to the ribozyme tested and moresequences need to be tested before a conclusion can be reachedconcerning the nuclease sensitivity of these sites, but the positions 7,8, 9 and 13 are conserved nucleotides in this motif and appear to besites at which nuclease digestion stops, as demonstrated by theappropriate stable fragments produced in lysate digestion experiments.Interestingly, although there seems to be much more nuclease in thelymphocyte and monocyte lysates, no stable fragments were observed whensimilar experiments were performed using an activated HeLa cytoplasmiclysate. Observations of ribozyme efficacy in HeLa CD4⁺ cells should bere-examined in lymphocytes before the therapeutic relevance can bedetermined.

EXAMPLE 1

HIV tat Ribozymes

The 5' exon of tat contains the following potential cleavage sites: 2GUC sites, 3 GUA sites, 5 AUC sites, 3 UCC sites, and 5 CUC sites. All18 sites were examined by computer folding and by RNaseH cleavage assay.

A measure of the accessibility of each site to binding by a 13-meroligonucleotide was preferred in the following way:

(a) The first 425 nucleotides of the clone V sequence (this clone wasmade available by Dr. Rossi and includes the 5' tat exon at nucleotides151-366) was folded on RNAFOLD 4.0 (a generally available program) andexamined for the presence of folding domains, i.e., self-containedstructures closed by a stem.

(b) For each potential cleavage site, the domain containing that sitewas folded to confirm that it folded as in part (a); then the domain wasrefolded while forcing the cleavage site and surrounding nucleotides(11-16 nucleotides in all) to remain unpaired. The difference in thesetwo folding free energies was taken as the cost of melting out thatregion for base-pairing by a ribozyme or DNA oligonucleotide.

(c) The lengths of DNA oligonucleotides were adjusted to give predicteddelta-G (binding) of -17 to -18 kcal/mole. Thus, differences in overallbinding energy was predicted to be reflected in the free energydifferences calculated in part (b). The calculations are shown in Table5.

                  TABLE 5                                                         ______________________________________                                        Total                Delta G       Delta G                                    Start                Melting       Binding                                                                              Delta G                             Seq         # Site   Structure                                                                            Length Oligo  Binding                             ______________________________________                                        194  GUC     1 (@200)                                                                              +8.6   11     -17.2  -8.6                                332  GUC     2 (@338)                                                                              +8.2   11     -17.3  -9.1                                156  GUA     3 (@163)                                                                              +7.5   12     -17.7  -10.2                               211  GUA     4 (@218)                                                                              +8.0   13     -17.5  -9.7                                225  GUA     5 (@233)                                                                              +6.8   15     -16.3  -9.5                                161  AUC     6 (@167)                                                                              +5.4   13     -18.0  -12.6                               186  AUC     7 (@192)                                                                              +1.6   11     -17.7  -16.1                               280  AUC     8 (@286)                                                                              +5.5   11     -17.9  -12.4                               340  AUC     9 (@347)                                                                              +7.1   13     -17.0  -9.9                                352  AUC    10 (@360)                                                                              +1.7   14     -17.4  -15.7                               240  UUC    11 (@248)                                                                              +11.7  14     -17.4  -5.7                                257  UUC    12 (@265)                                                                              +10.7  16     -17.4  -6.7                                346  UUC    13 (@354)                                                                              +9.7   14     -17.2  -7.5                                281  CUC    14 (@288)                                                                              +5.5   12     -17.6  -12.1                               319  CUC    15 (@326)                                                                              +11.9  11     -17.1  -5.2                                322  CUC    16 (@329)                                                                              +13.8  11     -17.5  -3.7                                337  CUC    17 (@344)                                                                              +6.3   12     -17.2  -10.9                               349  CUC    18 (@356)                                                                              +10.6  14     -17.2  -6.6                                ______________________________________                                         [site refers to nucleotide 5' of cleaved phosphate                       

Eighteen DNA oligonucleotides were made to target the 18 target siteslisted in Table 5. RNaseH experiments were performed using approximately100 nM body labeled RNA transcript (clone V), 0.08 U/μl RNaseH (excessRNaseH) and a 2× dilution of 1 mM, 10 μM or 1 μM DNA oligonucleotide.The results are shown in Table 5. Eight out of 18 sites had greater than40% cleavage at 5 μM after 10 minutes incubation. Three ribozymes weredesigned against the three most active sequences (H332, H337b, H352, seeTable 6).

                                      TABLE 6                                     __________________________________________________________________________                                 ENERGIES     % CLEAVAGE                          Oligo Name                                                                          Sequence (5'-->3')                                                                        Core                                                                             Length                                                                            Binding                                                                           Melting                                                                           Net 1 mM/30*                                                                           10 μM/10*                                                                        1 μM/10*                   __________________________________________________________________________    H156  GATCTACTGGCT                                                                              GUA                                                                              12  -18.5                                                                             7.5 -11 83   36    0                             H161  GTCTAGGATCTAC                                                                             AUC                                                                              13  -18.8                                                                             5.4 -13.4                                                                             12   0     0                             H186  TCCTGGATGCT AUC                                                                              11  -18.3                                                                             9.4 -8.9                                                                              82   0     0                             H194b GGCTGACTTCC GUC                                                                              11  -18.6                                                                             8.6 -10 99   84    0                             H211v2                                                                              ATTGGTACAAGCA                                                                             GUA                                                                              13  -18 8   -10 61   21    0                             H225  ACTTTTTACAATAGC                                                                           GUA                                                                              15  -17 6.8 -10.2                                                                             47   0     0                             H240  GCAATGAAAGCAAC                                                                            UUC                                                                              13  -19.4                                                                             11.7                                                                              -7.7                                                                              25   0     0                             H257  TTGTTATGAAACAAAC                                                                          UUC                                                                              16  -18 10.7                                                                              -7.3                                                                              100  0     0                             H280  AGGAGATGCCT AUC                                                                              11  -18.2                                                                             5.5 -12.7                                                                             96   60    0                             H281  ATAGGAGATGCC                                                                              CUC                                                                              12  -18.5                                                                             6.3 -12.2                                                                             84   11    0                             H319  GAGGAGGTCTT CUC                                                                              11  -17.4                                                                             11.9                                                                              -5.5                                                                              87   40    0                             H322  CTTGAGGAGGT CUC                                                                              11  -16.9                                                                             13.,8                                                                             -3.1                                                                              94   8     0                             H332  GTCTGACTGCC GUC                                                                              11  -18.7                                                                             8.2 -10.5                                                                             94   88    0                             H337b TGATGAGTCTGA                                                                              CUC                                                                              12  -17.6                                                                             6.3 -11.3                                                                             100  99    80                            H340v2                                                                              AACTTGATGAGTC                                                                             AUC                                                                              13  -17.4                                                                             7.1 -10.3                                                                             99   66    0                             H346  ATAGAGAAACTTGA                                                                            UUC                                                                              14  -17.2                                                                             9.7 -7.5                                                                              69   10    0                             H349  TTGATAGAGAAACT                                                                            CUC                                                                              14  -17.2                                                                             10.6                                                                              -6.6                                                                              55   23    0                             H352  GCTTTGATAGAGAA                                                                            AUC                                                                              14  -18.5                                                                             1.7 -16.8                                                                             94   100   84                            __________________________________________________________________________

These three ribozymes are shown in FIGS. 3A, B and C, labeledrespectively HDH, HEH and HFH.

EXAMPLE 2

Ribozymes Containing Thiophosphate

The purpose of this example was to evaluate the activity of ribozymescontaining substitutions of thiophosphate for phosphate at some backbonepositions.

Ribozymes to the HIV-1 tat gene were synthesized on an ABI synthesizerusing standard phosphoramidite chemistry, however, at steps wherethiophosphate was to be incorporated in the backbone the standardoxidation step (involving Iodine) was replaced with an oxidation steputilizing the Beaucage sulfur transfer reagent. Deprotected and desaltedRNA was gel purified, eluted, kinased and sequenced to ensure that thesequence was correct. The end-labeled RNA was also treated with H₂ O₂which preferentially promotes cleavage at positions containingthiophosphate.

Ribozyme activity was tested against cleavage of a short (12nucleotides) end-labeled substrate RNA. Substrate concentration wasapproximately 1 nM; ribozyme concentration was 5-100 nM; incubation wasat 37° C. in 75 mM Tris (pH 7.5), 0.1 mM EDTA, 10 mM MgCl₂ for 2-40minutes. Cleavage extents were determined by gel electrophoresis (PAGE)followed by quantitation on the AMBIS.

The ribozymes are shown in FIGS. 4A-4G. The following ribozymes showedessentially 100% activity: r37 (unmodified), r37s2 (1 thio on 5' arm, 2on 3' arm), r37s4 (3 thio modifications on each arm), s37A (3modifications on 3' arm), s37B (3 modifications alternating on 3' arm).

Ribozymes that are fully modified showed some decrease in activity, forexample, s37C (almost completely modified on substrate binding arms)showed a 3× reduction in activity relative to unmodified, s37D (thiomodifications in stem I, II and III) showed about a 9× reduction inactivity relative to unmodified.

EXAMPLE 3

2'-O-methyl-containing Ribozymes

Ribozymes were made on an ABI synthesizer using standard phosphoramiditechemistry with 2'-O-methyl phosphoramidite nucleotides used in place ofstandard nucleotides. Ribozyme activity was determined in the mannerdescribed above with PAGE analysis.

Referring to FIG. 5, a ribozyme containing 2'-O-methyl at all positionsthat base-pair with substrate (except for the A at the 5' side of stemIII) was synthesized. The Kcat/Km for the 2'-O-methyl ribozyme was44×10⁶ M⁻¹ min⁻¹ compared to 41×10⁶ m⁻¹ min⁻¹ for the unmodified, and32×10⁶ for a thiophosphate modified ribozyme. Thus, the 2'-O-methylribozyme retains 100% activity.

EXAMPLE 4

Targeting the LTR and TAT Regions of HIV-1

The following example extends those provided above to show usefulribozymes targeted to the LTR and TAT regions of HIV-1. Details ofmethodology used herein are provided in Stinchcomb et al., Methods andCompositions for Treatment of Restenosis and Cancer Using Ribozymes,U.S. Ser. No. 08/245,466, filed May 18, 1994 hereby incorporated byreference herein. Such details are not required to practice theinvention. Numbering of bases is according to GenBank Nos. K03455(HIVHXB2) (numbered from transcription start site).

Screening LTR region for HH Ribozyme Sites

The LTR is among the most conserved regions within the HIV-1 genome.Also, the LTR region is present in all the transcripts generated duringHIV-1 life cycle. So, ribozymes that cleave LTR targets will potentiallyblock HIV replication.

There are 43 potential hammerhead (HH) ribozyme sites within the HIV-1LTR. Ten hammerhead ribozymes were synthesized based on a) properfolding of the ribozyme with its target and b) conservation of targetsequence among all HIV-1 strains.

RNA Synthesis

Ribozymes with 7/7 binding arms were synthesized using RNAphosphoramadite chemistry. Ribozymes were deprotected and purified asdescribed above.

Target RNA used in this study was 613 nt long and contained cleavagesites for all the 10 HH ribozymes targeted against LTR. A templatecontaining T7 RNA polymerase promoter upstream of LTR target sequence,was PCR amplified from an HIV-1 pro-DNA clone. Other such clones can bereadily constructed. Target RNA was transcribed from this PCR amplifiedtemplate using T7 RNA polymerase. The transcript was internally labeledduring transcription by including [α-³² P] CTP as one of the fourribonucleotide triphosphates. The transcription mixture was treated withDNase-1, following transcription at 37° C. for 2 hours, to digest awaythe DNA template used in the transcription. RNA was precipitated withisopropanol and the pellet was washed two times with 70% ethanol to getrid of salt and nucleotides used in the transcription reaction. RNA isresuspended in DEPC-treated water and stored at 4° C.

Ribozyme Cleavage Reactions

Reactions were carried out under ribozyme excess (_(cat) /K_(m))conditions (Herschlag and Cech (1990) Biochemistry 29, 10159-10171).Briefly, 1,000 nM ribozyme and 10 nM internally labeled target RNA weredenatured separately by heating to 90° C. for 2 min in the presence of50 mM Tris.HCl, pH 7.5 and 10 mM MgCl₂. The RNAs were renatured bycooling to 37° C. for 10-20 min. Cleavage reaction was initiated bymixing the ribozyme and target RNA at 37° C. Aliquots of 5 μl were takenat regular intervals of time and the reaction was quenched by addingequal volume of stop buffer and freezing on dry ice. The samples wereresolved on sequencing gel.

Results

As shown in FIG. 10, of the ten ribozymes that were tested individually,only four HH ribozymes (568, 581, 631, 696) cleaved the LTR RNA. Othertarget sites appear to be inaccessible to ribozyme binding and cleavage.Site 568 is also referred to as site 115 and has been targeted forhammerhead ribozyme cleavage by Drouplic et al., 66 J. Virol. 1432-1441,1992 and Heidenreich and Eckstein, 267 J. Biol. Chem. 1904-1909, 1992.

Since 568 HH ribozyme was cleaving its target to a greater extent thanthe others, we were interested in optimizing the length of binding armsof this HH ribozyme. As shown in FIG. 11, the rate of ribozyme cleavageincreased significantly when the length of the binding arm was increasedfrom 12 to 14 base pairs (total). There was no significant improvementin the activity of ribozymes with binding arms longer than 14 basepairs.

Chemical modification of HH ribozymes targeted to a specific site cansignificantly improve the stability of the ribozyme in human serum.Further, these modifications do not seem to have any significant effecton the catalytic activity of the ribozyme. All the 2' hydroxyl groupswithin the ribozyme, with the exception of positions U4, G5, A6, U7, G8,G12 and A15.1 (using standard nomenclature, See FIG. 14), were modifiedwith 2'-O-methyl groups. The 2' hydroxyl groups at U4 and U7 weremodified with either 2'amino, 2'-C-allyl, 2'-O-methyl or 2'ara-flouro.See Usman et al., 2'-Deoxy 2'-alkylnucleotide containing Nucleic Acid,U.S. Ser. No. 08/218,934, filed Mar. 29, 1994, hereby incorporated byreference herein.

Referring to FIG. 12, the 568 HH ribozyme could be stabilized bychemical modification of the sugar moeity of various bases (similar tothe ones listed above). The 568 HH ribozyme was extensively modifiedwith one of the following compounds: 2'flouro, 2'amino at the U7position, araflouro at U4 position, 2'amino at U4 and U7 positions,c-allyl at U4 position. None of the above modifications had anydeleterious effect on the ribozyme activity.

Transfection of cells with 568 HH ribozyme (with U4 and U7 positionscontaining 2'amino modifications) blocks the replication of HIV-1replication.

Screening TAT Region for HH Ribozyme Sites

A region of the TAT mRNA is present in the majority of the transcriptsgenerated during HIV-1 life cycle. So, ribozymes that cleave TAT targetswill potentially block HIV replication.

There are 54 potential HH ribozyme site within the HIV-1 TAT regions(between 5776 nt and 6044 nt). Nine hammerhead ribozymes weresynthesized based on a) proper folding of the ribozyme with its targetand b) conservation of target sequence among all HIV-1 strains.

RNA synthesis and ribozyme cleavage reactions were carried out asdescribed above. Target RNA used in this study was 422 nt long.

As shown in FIG. 13, five sites (5841, 5869, 5878, 5966, 5969) arecleaved more readily than the others. None of these hammerhead siteshave previously been targeted.

Screening HIV-1 Genome for Hairpin Ribozyme Sites

Referring to Table VIII and FIG. 15, there are 27 potential hairpin (HP)ribozyme sites in the HIV-1 genome. Ribozymes shown in the table weresynthesized and tested. Modifications to various regions of the hairpinstructure can be made without deleterious effect, e.g., in thosetargetted to n.t. positions 565 and 4398 two extra bases can be insertedin place of GGCA (3rd col.) to GGCACA; and GCAG to GCAGUC respectively.

Site 565 within the LTR region (Ojwang et al., (1992) PNAS. USA. 89,10802-10806; Yu et al., (1993) PNAS. USA. 90, 6340-6344) and 4398 withinthe POL region of the HIV-1 genome (Joseph and Burke (1993) J. Biol.Chem. 268, 24515-24518) have been shown to be accessible to HP ribozymebinding and cleavage. We have also found that HP ribozymes targetedtowards 565 and 4398 sites are active.

The best HH and HP ribozymes are shown in Table VII, with theirassociated cleavage and target sites.

Administration of Ribozyme

Selected ribozymes can be administered prophylactically, or to HIV-1infected patients, e.g., by exogenous delivery of the ribozyme to aninfected tissue by means of an appropriate delivery vehicle, e.g., aliposome, a controlled release vehicle, by use of iontophoresis,electroporation or ion paired molecules, or covalently attached adducts,and other pharmacologically approved methods of delivery. Routes ofadministration include intramuscular, aerosol, oral (tablet or pillform), topical, systemic, ocular, intraperitoneal and/or intrathecal.Expression vectors for immunization with ribozymes and/or delivery ofribozymes are also suitable.

The specific delivery route of any selected ribozyme will depend on theuse of the ribozyme. Generally, a specific delivery program for eachribozyme will focus on unmodified ribozyme uptake with regard tointracellular localization, followed by demonstration of efficacy.Alternatively, delivery to these same cells in an organ or tissue of ananimal can be pursued. Uptake studies will include uptake assays toevaluate cellular ribozyme uptake, regardless of the delivery vehicle orstrategy. Such assays will also determine the intracellular localizationof the ribozyme following uptake, ultimately establishing therequirements for maintenance of steady-state concentrations within thecellular compartment containing the target sequence (nucleus and/orcytoplasm). Efficacy and cytotoxicity can then be tested. Toxicity willnot only include cell viability but also cell function.

Some methods of delivery that may be used include:

a. encapsulation in liposomes,

b. transduction by retroviral vectors,

c. conjugation with cholesterol,

d. localization to nuclear compartment utilizing antigen binding ornuclear targeting site found on most snRNAs or nuclear proteins,

e. neutralization of charge of ribozyme by using nucleotide derivatives,and

f. use of blood stem cells to distribute ribozymes throughout the body.

At least three types of delivery strategies are useful in the presentinvention, including: ribozyme modifications, particle carrier drugdelivery vehicles, and retroviral expression vectors. Unmodifiedribozymes, like most small molecules, are taken up by cells, albeitslowly. To enhance cellular uptake, the ribozyme may be modifiedessentially at random, in ways which reduce its charge but maintainsspecific functional groups. This results in a molecule which is able todiffuse across the cell membrane, thus removing the permeabilitybarrier.

Modification of ribozymes to reduce charge is just one approach toenhance the cellular uptake of these larger molecules. The randomapproach, however, is not advisable since ribozymes are structurally andfunctionally more complex than small drug molecules. The structuralrequirements necessary to maintain ribozyme catalytic activity are wellunderstood by those in the art. These requirements are taken intoconsideration when designing modifications to enhance cellular delivery.The modifications are also designed to reduce susceptibility to nucleasedegradation. Both of these characteristics should greatly improve theefficacy of the ribozyme. Cellular uptake can be increased by severalorders of magnitude without having to alter the phosphodiester linkagesnecessary for ribozyme cleavage activity.

Chemical modifications of the phosphate backbone will reduce thenegative charge allowing free diffusion across the membrane. Thisprinciple has been successfully demonstrated for antisense DNAtechnology. The similarities in chemical composition between DNA and RNAmake this a feasible approach. In the body, maintenance of an externalconcentration will be necessary to drive the diffusion of the modifiedribozyme into the cells of the tissue. Administration routes which allowthe diseased tissue to be exposed to a transient high concentration ofthe drug, which is slowly dissipated by systemic adsorption arepreferred. Intravenous administration with a drug carrier designed toincrease the circulation half-life of the ribozyme can be used. The sizeand composition of the drug carrier restricts rapid clearance from theblood stream. The carrier, made to accumulate at the site of infection,can protect the ribozyme from degradative processes.

Drug delivery vehicles are effective for both systemic and topicaladministration. They can be designed to serve as a slow releasereservoir, or to deliver their contents directly to the target cell. Anadvantage of using direct delivery drug vehicles is that multiplemolecules are delivered per uptake. Such vehicles have been shown toincrease the circulation half-life of drugs which would otherwise berapidly cleared from the blood stream. Some examples of such specializeddrug delivery vehicles which fall into this category are liposomes,hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres.

From this category of delivery systems, liposomes are preferred.Liposomes increase intracellular stability, increase uptake efficiencyand improve biological activity.

Liposomes are hollow spherical vesicles composed of lipids arranged in asimilar fashion as those lipids which make up the cell membrane. Theyhave an internal aqueous space for entrapping water soluble compoundsand range in size from 0.05 to several microns in diameter. Severalstudies have shown that liposomes can deliver RNA to cells and that theRNA remains biologically active.

For example, a liposome delivery vehicle originally designed as aresearch tool, Lipofectin, has been shown to deliver intact mRNAmolecules to cells yielding production of the corresponding protein. Inanother study, an antibody targeted liposome delivery system containingan RNA molecule 3,500 nucleotides in length and antisense to astructural protein of HIV, inhibited virus proliferation in a sequencespecific manner. Not only did the antibody target the liposomes to theinfected cells, but it also triggered the internalization of theliposomes by the infected cells. Triggering the endocytosis is usefulfor viral inhibition. Finally, liposome delivered synthetic ribozymeshave been shown to concentrate in the nucleus of H9 (an example of anHIV-sensitive cell) cells and are functional as evidenced by theirintracellular cleavage of the sequence. Liposome delivery to other celltypes using smaller ribozymes (less than 142 nucleotides in length)exhibit different intracellular localizations.

Liposomes offer several advantages: They are non-toxic and biodegradablein composition; they display long circulation half-lives; andrecognition molecules can be readily attached to their surface fortargeting to tissues. Finally, cost effective manufacture ofliposome-based pharmaceuticals, either in a liquid suspension orlyophilized product, has demonstrated the viability of this technologyas an acceptable drug delivery system.

Other controlled release drug delivery systems, such as nonoparticlesand hydrogels may be potential delivery vehicles for a ribozyme. Thesecarriers have been developed for chemotherapeutic agents andprotein-based pharmaceuticals, and consequently, can be adapted forribozyme delivery.

Topical administration of ribozymes is advantageous since it allowslocalized concentration at the site of administration with minimalsystemic adsorption. This simplifies the delivery strategy of theribozyme to the disease site and reduces the extent of toxicologicalcharacterization. Furthermore, the amount of material to be applied isfar less than that required for other administration routes. Effectivedelivery requires the ribozyme to diffuse into the infected cells.Chemical modification of the ribozyme to neutralize negative charge maybe all that is required for penetration. However, in the event thatcharge neutralization is insufficient, the modified ribozyme can beco-formulated with permeability enhancers, such as Azone or oleic acid,in a liposome. The liposomes can either represent a slow releasepresentation vehicle in which the modified ribozyme and permeabilityenhancer transfer from the liposome into the infected cell, or theliposome phospholipids can participate directly with the modifiedribozyme and permeability enhancer in facilitating cellular delivery. Insome cases, both the ribozyme and permeability enhancer can beformulated into a suppository formulation for slow release.

Ribozymes may also be systemically administered. Systemic absorptionrefers to the accumulation of drugs in the blood stream followed bydistribution throughout the entire body. Administration routes whichlead to systemic absorption include: intravenous, subcutaneous,intraperitoneal, intranasal, intrathecal and ophthalmic. Each of theseadministration routes expose the ribozyme to an accessible diseasedtissue. Subcutaneous administration drains into a localized lymph nodewhich proceeds through the lymphatic network into the circulation. Therate of entry into the circulation has been shown to be a function ofmolecular weight or size. The use of a liposome or other drug carrierlocalizes the ribozyme at the lymph node. The ribozyme can be modifiedto diffuse into the cell, or the liposome can directly participate inthe delivery of either the unmodified or modified ribozyme to the cell.This method is particularly useful for treating AIDS using anti-HIVribozymes of this invention.

Also preferred in AIDS therapy is the use of a liposome formulationwhich can deliver oligonucleotides to lymphocytes and macrophages. Thisoligonucleotide delivery system inhibits HIV proliferation in infectedprimary immune cells. Whole blood studies show that the formulation istaken up by 90% of the lymphocytes after 8 hours at 37° C. Preliminarybiodistribution and pharmacokinetic studies yielded 70% of the injecteddose/gm of tissue in the spleen after one hour following intravenousadministration. This formulation offers an excellent delivery vehiclefor anti-AIDS ribozymes for two reasons. First, T-helper lymphocytes andmacrophages are the primary cells infected by the virus, and second, asubcutaneous administration delivers the ribozymes to the residentHIV-infected lymphocytes and macrophages in the lymph node. Theliposomes then exit the lymphatic system, enter the circulation, andaccumulate in the spleen, where the ribozyme is delivered to theresident lymphocytes and macrophages.

Intraperitoneal administration also leads to entry into the circulation,with once again, the molecular weight or size of the ribozyme-deliveryvehicle complex controlling the rate of entry.

Liposomes injected intravenously show accumulation in the liver, lungand spleen. The composition and size can be adjusted so that thisaccumulation represents 30% to 40% of the injected dose. The remainingdose circulates in the blood stream for up to 24 hours.

The chosen method of delivery should result in cytoplasmic accumulationin the afflicted cells and molecules should have somenuclease-resistance for optimal dosing. Nuclear delivery may be used butis less preferable. Most preferred delivery methods include liposomes(10-400 nm), hydrogels, controlled-release polymers, microinjection orelectroporation (for ex vivo treatments) and other pharmaceuticallyapplicable vehicles. The dosage will depend upon the disease indicationand the route of administration but should be between 100-200 mg/kg ofbody weight/day. The duration of treatment will extend through thecourse of the disease symptoms, usually at least 14-16 days and possiblycontinuously. Multiple daily doses are anticipated for topicalapplications, ocular applications and vaginal applications. The numberof doses will depend upon disease delivery vehicle and efficacy datafrom clinical trials.

Establishment of therapeutic levels of ribozyme within the cell isdependent upon the rate of uptake and degradation. Decreasing the degreeof degradation will prolong the intracellular half-life of the ribozyme.Thus, chemically modified ribozymes, e.g., with modification of thephosphate backbone, or capping of the 5' and 3' ends of the ribozymewith nucleotide analogues may require different dosaging. Descriptionsof useful systems are provided in the art cited above, all of which ishereby incorporated by reference herein.

The claimed ribozymes are also useful as diagnostic tools tospecifically or non-specifically detect the presence of a target RNA ina sample. That is, the target RNA, if present in the sample, will bespecifically cleaved by the ribozyme, and thus can be readily andspecifically detected as smaller RNA species. The presence of suchsmaller RNA species is indicative of the presence of the target RNA inthe sample.

Other embodiments are within the following claims.

                                      TABLE VII                                   __________________________________________________________________________    HIV (Strain HXB-2) Ribozyme and Target Sequences that Have Been Screened      and Tested In Vitro                                                           __________________________________________________________________________       nt.                                                                        Type                                                                             Position                                                                           Hairpin Ribozyme Sequence                                                                                         Substrate Sequence                __________________________________________________________________________    HP   121                                                                              CACCAUCCAAAG AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                         CCACU GAC CUUUGGAUGGUG          HP   515                                                                              UAUUGAGGCUUA AAGA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                         CCACU GCU UAAGCCUCAAUA          HP   565                                                                              AGUCACACAACA AGAA GGCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                         UGCCC GUC UGUUGUGUGACU          HP   565                                                                              AGUCACACAACA AGAAGGCACAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                         UGCCC GUC UGUUGUGUGACU          HP  2025                                                                                 UUCCACAUUUCC AAGA GCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                        GGGCU GUU GGAAAUGUGGAA        HP  4398                                                                                 GCCAUAUUCCUG AGAAGCAGUCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                        CUGUA GUC CAGGAAUAUGGC        HP  4398                                                                                 GCCAUAUUCCUG AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                        CUGUA GUC CAGGAAUAUGGC        HP  9205                                                                                 CACCAUCGAAAG AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                        CCACU GAC CUUUGGAUGGUG        HP  9599                                                                                 UAUUGAGGCUUA AAGA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                        CCACU GCU UAAGCCUCAAUA        HP  9649                                                                                 AGUCACACAACA AGAA GGCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                        UGCCC GUC                     __________________________________________________________________________                                                UGUUGUGUGACU                          nt.                                                                       Type                                                                             Position                                                                           Hammerhead Ribozyme Sequence                                                                                      Substrate Sequence                __________________________________________________________________________    HH   520                                                                                 UUUAUUGAGGCU CUGAUGAGGCCGAAAGGCCGAA AAGCAGUGGGUU                                                                         AACCCACUGCUUA                                                       AGCCUCAAUAAA                      HH   526                                                                                 GCAAGCUUUAUU CUGAUGAGGCCGAAAGGCCGAA AGGCUUAAGCAG                                                                         CUGCUUAAGCCUC                                                       AAUAAAGCUUGC                      HH   530                                                                                 CAAGGCAAGCUU CUGAUGAGGCCGAAAGGCCGAA AUUGAGGCUUAA                                                                         UUAAGCCUCAAUA                                                       AAGCUUGCCUUG                      HH   541                                                                                 CUUGAAGCACUC CUGAUGAGGCCGAAAGGCCGAA AGGCAAGCUUUA                                                                         UAAAGCUUGCCUU                                                       GAGUGCUUCAAG                      HH   555                                                                                 ACGGGCACACAC CUGAUGAGGCCGAAAGGCCGAA ACUUGAAGCACU                                                                         AGUGCUUCAAGUA                                                       GUGUGUGCCCGU                      HH   568                                                                                 AGUCACACAACA CUGAUGAGGCCGAAAGGCCGAA ACGGGCACACAC                                                                         GUGUGUGCCCGUC                                                       UGUUGUGUGACU                      HH   581                                                                                 CUCUAGUUACCA CUGAUGAGGCCGAAAGGCCGAA AGUCACACAACA                                                                         UGUUGUGUGACUC                                                       UGGUAACUAGAG                      HH   596                                                                                 AAGGGUCUGAGG CUGAUGAGGCCGAAAGGCCGAA AUCUCUAGUUAC                                                                         GUAACUAGAGAUC                                                       CCUCAGACCCUU                      HH   609                                                                                 CCACACUGACUA CUGAUGAGGCCGAAAGGCCGAA AAGGGUCUGAGG                                                                         CCUCAGACCCUUU                                                       UAGUCAGUGUGG                      HH   631                                                                                 GGGCGCCACUGC CUGAUGAGGCCGAAAGGCCGAA AGAGAUUUUCCA                                                                         UGGAAAAUCUCUA                                                       GCAGUGGCGCCC                      HH   696                                                                                 CUUCAGCAAGCC CUGAUGAGGCCGAAAGGCCGAA AGUCCUGCGUCG                                                                         CGACGCAGGACUC                                                       GGCUUGCUGAAG                      HH   805                                                                                 CCGCUUAAUACU CUGAUGAGGCCGAAAGGCCGAA ACGCUCUCGCAC                                                                         GUGCGAGAGCGUC                                                       AGUAUUAAGCGG                      HH  2028                                                                                    UUCCACAUUUCC CUGAUGAGGCCGAAAGGCCGAA ACAGCCCUUUUU                                                                      AAAAAGGGCUGUU                                                       GGAAAUGUGGAA                      HH  4398                                                                                    AUAUUCCUGGAC CUGAUGAGGCCGAAAGGCCGAA ACAGUCUACUUG                                                                      CAAGUAGACUGUA                                                       GUCCAGGAAUAU                      HH  5841                                                                                    UAGUCUAGGAUC CUGAUGAGGCCGAAAGGCCGAA ACUGGCUCCAUU                                                                      AAUGGAGCCAGUA                                                       GAUCCUAGACUA                      HH  5869                                                                                    GCUGACUUCCUG CUGAUGAGGCCGAAAGGCCGAA AUGCUUCCAGGG                                                                      CCCUGGAAGCAUC                                                       CAGGAAGUCAGC                      HH  5878                                                                                    CAGUUUUAGGCU CUGAUGAGGCCGAAAGGCCGAA ACUUCCUGGAUG                                                                      CAUCCAGGAAGUC                                                       AGCCUAAAACUG                      HH  5908                                                                                    AACACUUUUUAC CUGAUGAGGCCGAGAGGCCGAA AUAGCAAUUGGU                                                                      ACCAAUUGCUAUU                                                       GUAAAAAGUGUU                      HH  5911                                                                                 AGCAACACUUUU CUGAUGAGGCCGAAAGGCCGAA ACAAUAGCAAUU                                                                         AAUUGCUAUUGUA                                                       AAAAGUGUUGCU                      HH         GGCAAUGAAAGC CUGAUGAGGCCGAAAGGCCGAA ACACUUUUUACA                                                                         UGUAAAAAGUGUU                                                       GCUUUCAUUGCC                      HH         CCUGCCAUAGGA CUGAUGAGGCCGAAAGGCCGAA AUGCCUAAGGCU                                                                              AGCCUUAGCCAUC                                                  UCCUAUGGCAGG                      HH         UUCCUGCCAUAG CUGAUGAGGCCGAAAGGCCGAA AGAUGCCUAAGG                                                                              CCUUAGGCAUCUC                                                  CUAUGGCAGGAA                      HH         UUCUUCCUGCCA CUGAUGAGGCCGAAAGGCCGAA AGGAGAUGCCUA                                                                    UAGGCAUCUCCUA                                                            UGGGAGCAAGAA                      HH         UUUAUUGAGGCU CUGAUGAGGCCGAAAGGCCGAA AAGCAGUGGGUU                                                                    AACCCACUGUUUA                                                            AGCCUCAAUAAA                      HH         GCAAGCUUUAUU CUGAUGAGGCCGAAAGGCCGAA AGGCUUAAGCAG                                                                    CUGCUUAAGCCUC                                                            AAUAAAGCUUGC                      HH         CAAGACAAGCUU CUGAUGAGGCCGAAAGGCCGAA AUUGAGGCUUAA                                                                    UUAAGCCUCAAUA                                                            AAGCUUGCCUUG                      HH         CUUGAAGCACUC CUGAUGAGGCCGAAAGGCCGAA AGGCAAGCUUUA                                                                    UAAAGCUUGCCUU                                                            GAGUGCUUCAAG                      HH         ACGGGCACACAC CUGAUGAGGCCGAAAGGCCGAA ACUUGAAGCACU                                                                    AGUGCUUCAAGUA                                                            GUGUGUGCCCGU                      HH         AGUCACACAACA CUGAUGAGGCCGAAAGGCCGAA ACGGGCACACAC                                                                    GUGUGUGCCCGUC                                                            UGUUGUGUGACU                      HH         CUCUAGUUACCA CUGAUGAGGCCGAAAGGCCGAA AGUCACACAACA                                                                    UGUUGUGUGACUC                                                            UGGUAACUAGAG                      HH         AAGGGUCUGAGG CUGAUGAGGCCGAAAGGCCGAA AUCUCUAGUUAC                                                                    GUAACUAGAGAUC                                                            CCUCAGACCCUU                      HH         CCACACUGACUA CUGAUGAGGCCGAAAGGCCGAA AAGGGUCUGAGG                                                                    CCUCAGACCCUUU                                                            UAGUCAGUGUGG                      __________________________________________________________________________

                                      TABLE VIII                                  __________________________________________________________________________    HIV (Strain HXB2) Hairpin Ribozyme Sqequence                                  nt.                                                                           Position                                                                           Hairpin Ribozyme Sequence                                                                                          Substrate Sequence                  __________________________________________________________________________     121 CACCAUCCAAAG AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                        CCACU GAC CUUUGGAUGGUG               270             GAAAUGCUAGGC AGAA GUCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGG         UA                                            UGACA GCC                                                            GCCUAGCAUUUC                         273            GAUGAAAUGCUA AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGG         A                                             CAGCC GCC                                                            UAGCAUUUCAUC                         515         UAUUGAGGGUUA AAGA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGGA                                                         CCACU GCU                                                            UAAGCCUCAAUA                         565             AGUCACACAACA AGAA GGCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGG         UA                                            UGCCC GUC                                                            UGUUGUGUGACU                         565            AGUCACACAACA AGAAGGCACA ACCAGAGAAACACACGUUGUGGUACAUUACCUGG         UA                                            UGCCC GUC                                                            UGUUGUGUGACU                        2025            UUCCACAUUUCC AAGA GCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGU         A                                             GGGGU GUU                                                            GGAAAUGUGGAA                        3273    GGCAGCACUAUA AGAA GUAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              GUACA GCC                                                            UAUAGUGCUGCC                        3720    UCCUUUUGUAUG AGAA GUUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              AAACU GCC                                                            CAUACAAAAGGA                        4039    UGCAUAUUGUGA AGAA GUUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              UAACA GAC                                                            UCACAAUAUGCA                        4398    GCCAUAUUCCUG AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              CUGUA GUC                                                            CAGGAAUAUGGC                        4398    GCCAUAUUCCUG AGAAGCAGUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                            CUGUA GUC CAGGAAUAUGGC      4612    CGCCCACCAACA AGAA GCCC ACCAGAGAAACACACGUUGUGCUACAUUACCUGGGA                                                              GGGCC GCC                                                            UGUUGGUGGGCG                        5348    AUGAAUUAGUUG AGAA GCUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              UAGCA GAC                                                            CAACUAAUUCAU                        5379    CAGAGUCUGAAA AGAA GUCA ACCAGACAAACACACGUUGUGGUACAUUACCUGGUA                                                              UGACU GUU                                                            UUUCAGACUCUG                        5760    UGAAAAUGGAUA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGGA                                                              CUGCU GUU                                                            UAUCCAUUUUCA                        6334    UACCCCAUAAUA AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGGA                                                              UCACA GUC                                                            UAUUAUGGGGUA                        6454    UUGUGGGUUGGG AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGGA                                                              CCACA GAC                                                            CCCAACCCACAA                        7013    CUUCUUCUGCUA AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGGA                                                              UGGCA GUC                                                            UAGCAGAAGAAG                        7346    CUCCACAAUUAA AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              GCACA GUU                                                            UUAAUUGUGGAG                        7392    CAAGUACUAUUA AGAA GUUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              CAACU GUU                                                            UAAUAGUACUUG                        7819    CAGCGUCAUUGA AGAA GCGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              GCGCA GCC                                                            UCAAUGACGCUG                        7833    CUGGCCUGUACC AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              ACGGU GAC                                                            GGUACAGGCCAG                        7930    CUUGAUGCCCCA AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              UCACA GUC                                                            UGGGGCAUCAAG                        9205    CACCAUCCAAAG AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              CCACU GAC                                                            CUUUGGAUGGUG                        9354    GAAAUGCUAGGC AGAA GUCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              UGACA GCC                                                            GCCUAGCAUUUC                        9357    GAUGAAAUGCUA AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              CAGCC GCC                                                            UAGCAUUUCAUC                        9599    UAUUGAGGCUUA AAGA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              CCACU GCU                                                            UAAGCCUCAAUA                        9649    AGUCACACAACA AGAA GGCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA                                                              UGCCC GUC                  __________________________________________________________________________                                              UGUUGUGUGACU                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    #    232i) NUMBER OF SEQUENCES:                                               - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #    40 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #    40            UCAU UAGGGAUUAU GGAAAACAGA                                 - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                  #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (ix) FEATURE:                                                           #  The letter "N" stands for any                                                             base.                                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #    16 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #    28 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #             28   CUGG CAUUUGGG                                              - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #    23 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #                23CUCC AUA                                                   - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    30 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #           30     CACA AGUAGACCCU                                            - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    42 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #  42              CAUA ACAAGGUAGG AUCUCUACAA UA                              - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    44 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                # 44               GCAU UAAUAACACC AAAAAAGAUA AAGC                            - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    19 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                # 19               UAG                                                        - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #               25 UGAA ACUUA                                                 - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    13 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    14 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #     14                                                                      - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    26 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #              26  GUAA AAAGUG                                                - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    12 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    44 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                # 44               AAGC CUUAGGCAUC UCCUAUGGCA GGAA                            - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    14 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #     14                                                                      - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    26 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #              26  AAGU UUCUCU                                                - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    44 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                # 44               ACCC GACAGGCCCG AAGGAAUAGA AGAA                            - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    22 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                #                 22UGU GC                                                    - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    29 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                #            29    UUGG UGGAAUCUC                                             - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    12 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    16 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    23 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                #                23UUAU AGA                                                   - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    34 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                #        34        ACAG GGCUUGGAAA GGAU                                       - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    14 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                #     14                                                                      - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    22 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                #                 22AGC CA                                                    - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    28 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                #             28   GGAG CAAUCACA                                              - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    34 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                #        34        AGAG GAGGAGAAGG UGGG                                       - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    36 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                #       36         UAAG ACCAAUGACU UACAAG                                     - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    40 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                #    40            GCCA CUUUUUAAAA GAAAAGGGGG                                 - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    19 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                # 19               CGA                                                        - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    34 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                #        34        AUCU GUGGAUCUAC CACA                                       - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    24 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                #                24ACCA GGAC                                                  - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    14 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                #     14                                                                      - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    29 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                #            29    UGGA AUGGAUGAC                                             - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                # 20               CCGC                                                       - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    42 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                #  42              CUGA UAUCGAGCUU GCUACAAGGG AC                              - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    17 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    17 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    18 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                #  18              AC                                                         - (2) INFORMATION FOR SEQ ID NO:57:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    19 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                # 19               GCC                                                        - (2) INFORMATION FOR SEQ ID NO:58:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (ix) FEATURE:                                                           #  The letter "N" stands for any                                                             base.                                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:59:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (ix) FEATURE:                                                           #  The letter "R" stands for G or A.                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:60:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (ix) FEATURE:                                                           #  The letter "N" stands for any                                                             base.                                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:61:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (ix) FEATURE:                                                           #  The letter "W" stands for A or U.                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:62:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (ix) FEATURE:                                                           #  The letter "R" stands for A or G.                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:63:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (ix) FEATURE:                                                           #  The letter "Y" stands for U or C.                                                         The lette - #r "R" stands for G or A.                                         The lette - #r "N" stands for any                                             base.                                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:64:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:65:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:66:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:67:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:68:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:69:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                - CACCAUCCAA AGAGAAGUGG ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:70:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                - UAUUGAGGCU UAAAGAGUGG ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:71:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                - AGUCACACAA CAAGAAGGCA ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:72:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    58 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                - AGUCACACAA CAAGAAGGCA CAACCAGAGA AACACACGUU GUGGUACAUU AC - #CUGGUA           58                                                                          - (2) INFORMATION FOR SEQ ID NO:73:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                - UUCCACAUUU CCAAGAGCCC ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:74:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                - GCCAUAUUCC UGAGAAGCAG ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:75:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    58 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                - GCCAUAUUCC UGAGAAGCAG UCACCAGAGA AACACACGUU GUGGUACAUU AC - #CUGGUA           58                                                                          - (2) INFORMATION FOR SEQ ID NO:76:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                - CACCAUCCAA AGAGAAGUGG ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:77:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                - UAUUGAGGCU UAAAGAGUGG ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:78:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                - AGUCACACAA CAAGAAGGCA ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:79:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                # 20               GGUG                                                       - (2) INFORMATION FOR SEQ ID NO:80:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                # 20               AAUA                                                       - (2) INFORMATION FOR SEQ ID NO:81:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                # 20               GACU                                                       - (2) INFORMATION FOR SEQ ID NO:82:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    22 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                #                 22UGA CU                                                    - (2) INFORMATION FOR SEQ ID NO:83:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                # 20               GGAA                                                       - (2) INFORMATION FOR SEQ ID NO:84:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                # 20               UGGC                                                       - (2) INFORMATION FOR SEQ ID NO:85:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    22 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                #                 22AUG GC                                                    - (2) INFORMATION FOR SEQ ID NO:86:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                # 20               GGUG                                                       - (2) INFORMATION FOR SEQ ID NO:87:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                # 20               AAUA                                                       - (2) INFORMATION FOR SEQ ID NO:88:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                # 20               GACU                                                       - (2) INFORMATION FOR SEQ ID NO:89:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                #                 46GAG GCCGAAAGGC CGAAAAGCAG UGGGUU                          - (2) INFORMATION FOR SEQ ID NO:90:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                #                 46GAG GCCGAAAGGC CGAAAGGCUU AAGCAG                          - (2) INFORMATION FOR SEQ ID NO:91:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                #                 46GAG GCCGAAAGGC CGAAAUUGAG GCUUAA                          - (2) INFORMATION FOR SEQ ID NO:92:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                #                 46GAG GCCGAAAGGC CGAAAGGCAA GCUUUA                          - (2) INFORMATION FOR SEQ ID NO:93:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                #                 46GAG GCCGAAAGGC CGAAACUUGA AGCACU                          - (2) INFORMATION FOR SEQ ID NO:94:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                #                 46GAG GCCGAAAGGC CGAAACGGGC ACACAC                          - (2) INFORMATION FOR SEQ ID NO:95:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                #                 46GAG GCCGAAAGGC CGAAAGUCAC ACAACA                          - (2) INFORMATION FOR SEQ ID NO:96:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                #                 46GAG GCCGAAAGGC CGAAAUCUCU AGUUAC                          - (2) INFORMATION FOR SEQ ID NO:97:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                #                 46GAG GCCGAAAGGC CGAAAAGGGU CUGAGG                          - (2) INFORMATION FOR SEQ ID NO:98:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                #                 46GAG GCCGAAAGGC CGAAAGAGAU UUUCCA                          - (2) INFORMATION FOR SEQ ID NO:99:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                #                 46GAG GCCGAAAGGC CGAAAGUCCU GCGUCG                          - (2) INFORMATION FOR SEQ ID NO:100:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                               #                 46GAG GCCGAAAGGC CGAAACGCUC UCGCAC                          - (2) INFORMATION FOR SEQ ID NO:101:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                               #                 46GAG GCCGAAAGGC CGAAACAGCC CUUUUU                          - (2) INFORMATION FOR SEQ ID NO:102:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                               #                 46GAG GCCGAAAGGC CGAAACAGUC UACUUG                          - (2) INFORMATION FOR SEQ ID NO:103:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                               #                 46GAG GCCGAAAGGC CGAAACUGGC UCCAUU                          - (2) INFORMATION FOR SEQ ID NO:104:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                               #                 46GAG GCCGAAAGGC CGAAAUGCUU CCAGGG                          - (2) INFORMATION FOR SEQ ID NO:105:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:                               #                 46GAG GCCGAAAGGC CGAAACUUCC UGGAUG                          - (2) INFORMATION FOR SEQ ID NO:106:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:                               #                 46GAG GCCGAAAGGC CGAAAUAGCA AUUGGU                          - (2) INFORMATION FOR SEQ ID NO:107:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:                               #                 46GAG GCCGAAAGGC CGAAACAAUA GCAAUU                          - (2) INFORMATION FOR SEQ ID NO:108:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:                               #                 46GAG GCCGAAAGGC CGAAACACUU UUUACA                          - (2) INFORMATION FOR SEQ ID NO:109:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:                               #                 46GAG GCCGAAAGGC CGAAAUGCCU AAGGCU                          - (2) INFORMATION FOR SEQ ID NO:110:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:                               #                 46GAG GCCGAAAGGC CGAAAGAUGC CUAAGG                          - (2) INFORMATION FOR SEQ ID NO:111:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:                               #                 46GAG GCCGAAAGGC CGAAAGGAGA UGCCUA                          - (2) INFORMATION FOR SEQ ID NO:112:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:                               #                 46GAG GCCGAAAGGC CGAAAAGCAG UGGGUU                          - (2) INFORMATION FOR SEQ ID NO:113:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:                               #                 46GAG GCCGAAAGGC CGAAAGGCUU AAGCAG                          - (2) INFORMATION FOR SEQ ID NO:114:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:                               #                 46GAG GCCGAAAGGC CGAAAUUGAG GCUUAA                          - (2) INFORMATION FOR SEQ ID NO:115:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:                               #                 46GAG GCCGAAAGGC CGAAAGGCAA GCUUUA                          - (2) INFORMATION FOR SEQ ID NO:116:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:                               #                 46GAG GCCGAAAGGC CGAAACUUGA AGCACU                          - (2) INFORMATION FOR SEQ ID NO:117:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:                               #                 46GAG GCCGAAAGGC CGAAACGGGC ACACAC                          - (2) INFORMATION FOR SEQ ID NO:118:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:                               #                 46GAG GCCGAAAGGC CGAAAGUCAC ACAACA                          - (2) INFORMATION FOR SEQ ID NO:119:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:                               #                 46GAG GCCGAAAGGC CGAAAUCUCU AGUUAC                          - (2) INFORMATION FOR SEQ ID NO:120:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    46 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:                               #                 46GAG GCCGAAAGGC CGAAAAGGGU CUGAGG                          - (2) INFORMATION FOR SEQ ID NO:121:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:                               #               25 CUCA AUAAA                                                 - (2) INFORMATION FOR SEQ ID NO:122:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:                               #               25 AAAG CUUGC                                                 - (2) INFORMATION FOR SEQ ID NO:123:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:                               #               25 CUUG CCUUG                                                 - (2) INFORMATION FOR SEQ ID NO:124:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:                               #               25 UGCU UCAAG                                                 - (2) INFORMATION FOR SEQ ID NO:125:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:                               #               25 UGUG CCCGU                                                 - (2) INFORMATION FOR SEQ ID NO:126:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:                               #               25 UGUG UGACU                                                 - (2) INFORMATION FOR SEQ ID NO:127:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:                               #               25 UAAC UAGAG                                                 - (2) INFORMATION FOR SEQ ID NO:128:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:                               #               25 CAGA CCCUU                                                 - (2) INFORMATION FOR SEQ ID NO:129:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:                               #               25 UCAG UGUGG                                                 - (2) INFORMATION FOR SEQ ID NO:130:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:                               #               25 GUGG CGCCC                                                 - (2) INFORMATION FOR SEQ ID NO:131:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:                               #               25 UUGC UGAAG                                                 - (2) INFORMATION FOR SEQ ID NO:132:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:                               #               25 AUUA AGCGG                                                 - (2) INFORMATION FOR SEQ ID NO:133:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:                               #               25 AAUG UGGAA                                                 - (2) INFORMATION FOR SEQ ID NO:134:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:                               #               25 CAGG AAUAU                                                 - (2) INFORMATION FOR SEQ ID NO:135:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:                               #               25 CCUA GACUA                                                 - (2) INFORMATION FOR SEQ ID NO:136:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:                               #               25 GAAG UCAGC                                                 - (2) INFORMATION FOR SEQ ID NO:137:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:                               #               25 CUAA AACUG                                                 - (2) INFORMATION FOR SEQ ID NO:138:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:                               #               25 AAAA GUGUU                                                 - (2) INFORMATION FOR SEQ ID NO:139:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:                               #               25 AGUG UUGCU                                                 - (2) INFORMATION FOR SEQ ID NO:140:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:                               #               25 UUCA UUGCC                                                 - (2) INFORMATION FOR SEQ ID NO:141:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:                               #               25 UAUG GCAGG                                                 - (2) INFORMATION FOR SEQ ID NO:142:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:                               #               25 UGGC AGGAA                                                 - (2) INFORMATION FOR SEQ ID NO:143:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:                               #               25 CAGG AAGAA                                                 - (2) INFORMATION FOR SEQ ID NO:144:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:                               #               25 CUCA AUAAA                                                 - (2) INFORMATION FOR SEQ ID NO:145:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:                               #               25 AAAG CUUGC                                                 - (2) INFORMATION FOR SEQ ID NO:146:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:                               #               25 CUUG CCUUG                                                 - (2) INFORMATION FOR SEQ ID NO:147:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:                               #               25 UGCU UCAAG                                                 - (2) INFORMATION FOR SEQ ID NO:148:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:                               #               25 UGUG CCCGU                                                 - (2) INFORMATION FOR SEQ ID NO:149:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:                               #               25 UGUG UGACU                                                 - (2) INFORMATION FOR SEQ ID NO:150:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:                               #               25 UAAC UAGAG                                                 - (2) INFORMATION FOR SEQ ID NO:151:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:                               #               25 CAGA CCCUU                                                 - (2) INFORMATION FOR SEQ ID NO:152:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    25 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:                               #               25 UCAG UGUGG                                                 - (2) INFORMATION FOR SEQ ID NO:153:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:                               - GAAAUGCUAG GCAGAAGUCA ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:154:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154:                               - GAUGAAAUGC UAAGAAGCUG ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:155:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:                               - GGCAGCACUA UAAGAAGUAC ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:156:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:                               - UCCUUUUGUA UGAGAAGUUU ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:157:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:                               - UGCAUAUUGU GAAGAAGUUA ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:158:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:                               - CGCCCACCAA CAAGAAGCCC ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:159:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:                               - AUGAAUUAGU UGAGAAGCUA ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:160:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:                               - CAGAGUCUGA AAAGAAGUCA ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:161:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:                               - UGAAAAUGGA UAAGAAGCAG ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:162:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:                               - UACCCCAUAA UAAGAAGUGA ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:163:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:                               - UUGUGGGUUG GGAGAAGUGG ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:164:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:                               - CUUCUUCUGC UAAGAAGCCA ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:165:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:                               - CUCCACAAUU AAAGAAGUGC ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:166:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:                               - CAAGUACUAU UAAGAAGUUG ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:167:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:                               - CAGCGUCAUU GAAGAAGCGC ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:168:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168:                               - CUGGCCUGUA CCAGAAGCGU ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:169:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:                               - CUUGAUGCCC CAAGAAGUGA ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:170:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:                               - GAAAUGCUAG GCAGAAGUCA ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:171:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    56 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:                               - GAUGAAAUGC UAAGAAGCUG ACCAGAGAAA CACACGUUGU GGUACAUUAC CU - #GGUA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:172:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:                               # 20               UUUC                                                       - (2) INFORMATION FOR SEQ ID NO:173:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:                               # 20               CAUC                                                       - (2) INFORMATION FOR SEQ ID NO:174:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174:                               # 20               UGCC                                                       - (2) INFORMATION FOR SEQ ID NO:175:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:                               # 20               AGGA                                                       - (2) INFORMATION FOR SEQ ID NO:176:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:176:                               # 20               UGCA                                                       - (2) INFORMATION FOR SEQ ID NO:177:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:177:                               # 20               GGCG                                                       - (2) INFORMATION FOR SEQ ID NO:178:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:178:                               # 20               UCAU                                                       - (2) INFORMATION FOR SEQ ID NO:179:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:179:                               # 20               UCUG                                                       - (2) INFORMATION FOR SEQ ID NO:180:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:180:                               # 20               UUCA                                                       - (2) INFORMATION FOR SEQ ID NO:181:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:181:                               # 20               GGUA                                                       - (2) INFORMATION FOR SEQ ID NO:182:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:182:                               # 20               ACAA                                                       - (2) INFORMATION FOR SEQ ID NO:183:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:183:                               # 20               GAAG                                                       - (2) INFORMATION FOR SEQ ID NO:184:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:184:                               # 20               GGAG                                                       - (2) INFORMATION FOR SEQ ID NO:185:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:185:                               # 20               CUUG                                                       - (2) INFORMATION FOR SEQ ID NO:186:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:186:                               # 20               GCUG                                                       - (2) INFORMATION FOR SEQ ID NO:187:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:187:                               # 20               CCAG                                                       - (2) INFORMATION FOR SEQ ID NO:188:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:188:                               # 20               CAAG                                                       - (2) INFORMATION FOR SEQ ID NO:189:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:189:                               # 20               UUUC                                                       - (2) INFORMATION FOR SEQ ID NO:190:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:190:                               # 20               CAUC                                                       - (2) INFORMATION FOR SEQ ID NO:191:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:191:                               #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:192:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:192:                               #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:193:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:193:                               #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:194:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:194:                               #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:195:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    12 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:195:                               #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:196:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    13 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:196:                               #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:197:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:197:                               #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:198:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:198:                               #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:199:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    13 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:199:                               #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:200:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:200:                               #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:201:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    14 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:201:                               #     14                                                                      - (2) INFORMATION FOR SEQ ID NO:202:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    16 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:202:                               #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:203:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:203:                               #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:204:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    12 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:204:                               #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:205:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:205:                               #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:206:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:206:                               #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:207:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:207:                               #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:208:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    12 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:208:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:209:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    13 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:209:                               #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:210:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    14 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:210:                               #     14                                                                      - (2) INFORMATION FOR SEQ ID NO:211:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    14 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:211:                               #     14                                                                      - (2) INFORMATION FOR SEQ ID NO:212:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    14 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:212:                               #     14                                                                      - (2) INFORMATION FOR SEQ ID NO:213:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    13 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:213:                               #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:214:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    34 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:214:                               #        34        CGAA AGGCCGAAAC UGCC                                       - (2) INFORMATION FOR SEQ ID NO:215:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    13 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:215:                               #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:216:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    34 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:216:                               #        34        CGAA AGGCCGAAAG UCUG                                       - (2) INFORMATION FOR SEQ ID NO:217:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    13 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:217:                               #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:218:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    34 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:218:                               #        34        CGAA AGGCCGAAAU AGAG                                       - (2) INFORMATION FOR SEQ ID NO:219:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    14 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:219:                               #     14                                                                      - (2) INFORMATION FOR SEQ ID NO:220:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    37 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:220:                               #      37          CGCG AAAGCGGCGA AACUUCC                                    - (2) INFORMATION FOR SEQ ID NO:221:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    15 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:221:                               #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:222:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    36 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:222:                               #       36         CGAA AGGCCGAAAC CCCAAU                                     - (2) INFORMATION FOR SEQ ID NO:223:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    13 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:223:                               #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:224:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    36 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:224:                               #       36         CCGA AAGGACCGAA ACGGUC                                     - (2) INFORMATION FOR SEQ ID NO:225:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    11 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (ix) FEATURE:                                                           #  The letter "N" stands for any                                                             base. The - # letter "H" stands                                #U, or C.      for A,                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:225:                               #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:226:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    32 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (ix) FEATURE:                                                           #  The letter "N" stands for any                                                             base.                                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:226:                               #          32      NNNN NNNCGAAANN NN                                         - (2) INFORMATION FOR SEQ ID NO:227:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    20 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:227:                               # 20               UGGC                                                       - (2) INFORMATION FOR SEQ ID NO:228:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    38 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:228:                               #     38           GCAG ACCAGAGAAA CACACGCG                                   - (2) INFORMATION FOR SEQ ID NO:229:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    21 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:229:                               #21                UGGU A                                                     - (2) INFORMATION FOR SEQ ID NO:230:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    22 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:230:                               #                 22AUG GC                                                    - (2) INFORMATION FOR SEQ ID NO:231:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    40 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:231:                               #    40            GCAG UCACCAGAGA AACACACGCG                                 - (2) INFORMATION FOR SEQ ID NO:232:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #    21 base pairsTH:                                                         #      nucleic acid                                                           #single   (C) STRANDEDNESS:                                                   #   linear(D) TOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:232:                               #21                UGGU A                                                     __________________________________________________________________________

We claim:
 1. An enzymatic nucleic acid molecule which specificallycleaves RNA encoded by the nef gene of human immunodeficiency virus. 2.The enzymatic nucleic acid molecule of claim 1, wherein said enzymaticnucleic acid molecule is in a hammerhead motif.
 3. The enzymatic nucleicacid molecule of claim 1, wherein said enzymatic nucleic acid moleculeis in a hairpin motif.
 4. The enzymatic nucleic acid molecule of claim1, wherein said enzymatic nucleic acid molecule is in a group I intron,group II intron, hepatitis delta virus ribozyme or RNase P M1 RNA motif.5. The enzymatic nucleic acid molecule of claim 1, wherein saidenzymatic nucleic acid molecule comprises a substrate binding sitehaving between 12 and 100 nucleotides complementary to said nef RNA. 6.The enzymatic nucleic acid molecule of claim 1, wherein said enzymaticnucleic acid molecule comprises a substrate binding site having between14 and 24 nucleotides complementary to said nef RNA.
 7. An expressionvector comprising nucleic acid sequence encoding one or more enzymaticnucleic acid molecule of claim 1 in a manner which allows expression ofsaid enzymatic nucleic acid molecules.
 8. The expression vector of claim7, wherein said expression vector is a viral vector.
 9. The expressionvector of claim 8, wherein said viral vector is a retrovirus vector. 10.The enzymatic nucleic acid molecule of claim 1, wherein said enzymaticnucleic acid molecule is chemically synthesized.
 11. The enzymaticnucleic acid molecule of claim 1, wherein said enzymatic nucleicmolecule is in a purified form.
 12. The enzymatic nucleic acid moleculeof claim 1, wherein said enzymatic RNA molecule is active in thepresence of divalent metal ions.
 13. The enzymatic nucleic acid moleculeof claim 12, wherein said divalent metal ion is magnesium.
 14. Theenzymatic nucleic acid molecule of claim 1, wherein said enzymaticnucleic molecule comprises a sugar modification.
 15. The expressionvector of claim 7, wherein said nucleic acid sequence encoding saidenzymatic RNA molecule is under the control of a mammalian transcriptionpromoter.
 16. The expression vector of claim 15, wherein said mammaliantranscription promoter is from a cytomegalovirus.
 17. A method ofcleaving RNA encoded by the nef gene comprising the step of contactingsaid RNA with the enzymatic nucleic acid molecule of claim 1 underconditions suitable for the cleavage of said RNA.
 18. A mammalian cellincluding the enzymatic nucleic acid molecule of claim 1, wherein themammalian cell is not part of a mammal.
 19. The mammalian cell of claim18, wherein said mammalian cell is a human cell.
 20. A mammalian cellincluding the expression vector of claim
 7. 21. The mammalian cell ofclaim 20, wherein said mammalian cell is a human cell.
 22. The enzymaticnucleic acid molecule of claim 1, wherein said enzymatic nucleic acidmolecule comprises a poly A tail.